Apparatus and method for detection and discrimination molecular object

ABSTRACT

An apparatus for detecting an object capable of emitting light. The apparatus comprises a light detector comprising at least two optical sensors capable of determining the intensity of the light; and a computer processing output signal generated by the optical sensors and comparing a result of the processing with a known result corresponding to a known type to determine whether the object belongs to the known type.

RELATED APPLICATIONS

This application is a division of application Ser. No. 12/720,352, filedMar. 9, 2010, which claims the benefit of priority to U.S. ProvisionalPatent Application No. 61/159,310, filed Mar. 11, 2009, the entirecontent of all of which is incorporated by reference herein in itsentirety.

TECHNICAL FIELD

The present invention relates to a detecting apparatus, and the methodof using this apparatus to detect and/or discriminate an object. Furtherrelates to a detecting apparatus that is able to detect and/ordiscriminate an object emitting a light of low intensity.

BACKGROUND

The Human Genome Project (HGP) spurred a great increase in sequencingthroughput and resulted in a corresponding drop in sequencing costs. Incontrast to the 13 years and cost of nearly three billion US dollars,per genome sequencing costs have been reduced significantly—indeed twoindividual genomes have recently been completed (McGuire et al., Science317:1687 (2007)). Personal genomes represent a paradigm shift in medicaltreatment for both patients and health care providers. By managinggenetic risk factors for disease, health care providers can more readilypractice preventative medicine and provide customized treatment. Withlarge banks of completed genomes, drug design and administration can bemore efficient, pushing forward the nascent field of pharmacogenomics.

Many conventional DNA sequencing technologies implement optoelectronictechnique as a means to detect and/or discriminate an object bydetecting light emitted from the object. A detecting apparatuses used inthese technologies are often expensive and the efficiencies are nothigh.

Conventional methods are usually based on the measurement within acertain wavelength band where the light emitted from the object(s) beingdetected has the highest intensity. The measured intensity is then usedto calculate the concentration or amount of the objects. Recently,detecting light emitted from a single object is becoming popular. Forexample, one may need to detect the fluorescent light emitted from asingle dye molecule so as to, e.g., discriminate the molecule. Theintensity of such a fluorescent light can be very low, such thatconventional detecting apparatuses and methods are not suitable todetect such a weak light. Also, the sensitivity of analytical proceduressuch as flow cytometry and flow-cytometry-like microfluidiclab-on-a-chip device can be limited by the sensitivity of the lightdetector. Increasing the sensitivity of such procedures could allow themto detect materials present at relatively low levels which nonethelessmay be of interest in, for example, diagnostic or research applications.

Moreover, in many conventional apparatuses, color filters are oftenused, allowing a portion of the emitted light within a certainwavelength band to pass through and blocking other portion of theemitted light. Therefore, the apparatuses are complicated and more spaceis needed. Besides, since part of the emitted light is blocked by thecolor filter, the number of photons reaching the light detector isreduced. This makes such conventional apparatuses and methods even lesssuitable for detecting and/or discriminating, e.g., object with weakemission.

Therefore, there is a need for an apparatus and a method to detectand/or discriminate an object, especially an object emitting light oflow intensity such as a single dye molecule.

SUMMARY OF THE INVENTION

In accordance with the invention, there is provided an apparatus fordetecting an object capable of emitting light. The apparatus comprises alight detector comprising at least a first optical sensor and a secondoptical sensor capable of determining the intensity of the light; and acomputer processing output signal generated by the optical sensors andcomparing a result of the processing with a known result correspondingto a known type to determine whether the object belongs to the knowntype.

Also in accordance with the invention, there is provided a method fordetecting an object capable of emitting light. The method comprisesproviding a light detector comprising at least a first optical sensorand a second optical sensor, wherein the light detector absorbs lightemitted from the at least one object; processing output signal generatedby the at least two optical sensors; and comparing at least one resultof the processing with at least one known result corresponding to atleast one known type to determine whether the at least one objectbelongs to the at least one known type.

Also in accordance with the invention, there is provided anon-transitory computer-readable medium encoded with a computer programproduct. The computer program product, when executed by a computer,instructed the computer to process output signal generated by at least afirst optical sensor and a second optical sensor, wherein the signals isgenerated in response to absorbing light emitted from an object; and tocompare a result of the processing with a known result corresponding toa known type to determine whether an object belongs to the known type.

Also in accordance with the invention, there is provided a method ofsequencing a nucleic acid, comprising the steps of: (a) performingsingle molecule nucleic acid sequencing of the at least one nucleic acidmolecule, wherein the single molecule nucleic acid sequencing leads toemission of light correlated to the identity of at least one basecomprised by the nucleic acid; (b) detecting said light with at leastone light detector comprising at least a first optical sensor and asecond optical sensor; (c) processing signal output from the at leasttwo optical sensors; and (d) comparing at least one result of theprocessing with at least one known result corresponding to at least oneknown type to determine an identity of at least one base comprised bythe nucleic acid.

Additional objects and advantages of the invention will be set forth inpart in the description which follows, and in part will be obvious fromthe description, or may be learned by practice of the invention. Theobjects and advantages of the invention will be realized and attained bymeans of the elements and combinations particularly pointed out in theappended claims.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed.

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate one (several) embodiment(s) ofthe invention and together with the description, serve to explain theprinciples of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the absorption coefficient of silicon.

FIG. 2 is a view showing a detecting apparatus consistent with thepresent invention.

FIG. 3 is a view showing a light detector consistent with the presentinvention.

FIG. 4 is a view showing a light detector consistent with the presentinvention.

FIG. 5 is a view showing a grating used in a detecting apparatusconsistent with the present invention.

FIG. 6 is a graph showing the emission spectra of two quantum dots.

FIG. 7 is a graph showing the responsivity curves of two photodiodesconsistent with the present invention.

FIG. 8 is a graph showing the spectra of two quantum dots and theresponsivity curves of two photodiodes in one figure.

FIG. 9 is a flow chart illustrating a detecting method according to oneembodiment of the invention.

FIG. 10 is a flow chart illustrating a detecting method according toanother embodiment of the invention.

FIG. 11 is a flow chart illustrating a detecting method according tostill another embodiment of the invention.

FIG. 12 is a flow chart illustrating a detecting method according to afurther embodiment of the invention.

FIG. 13 is a flow chart illustrating a detecting method according to yetanother embodiment of the invention.

FIG. 14 is a flow chart illustrating a detecting method according to yeta further embodiment of the invention.

FIG. 15 is a flow chart illustrating a detecting method according tostill a further embodiment of the invention.

FIG. 16 is a view showing an example detecting apparatus consistent withthe present invention.

FIG. 17 is a view showing a multi-junction photodiode as an example ofthe light detector consistent with the present invention.

FIG. 18 is a graph showing the responsivity curve of one photodiode inthe multi-junction photodiode of FIG. 11.

FIG. 19 is a graph showing the responsivity curve of another photodiodein the multi-junction photodiode of FIG. 11.

FIG. 20 is a graph showing the absorption spectra of three objectstested in one example of the present invention.

FIG. 21 is a graph showing the emission spectra of three objects testedin one example of the present invention.

DETAILED DESCRIPTION

Embodiments consistent with the present invention include a detectingapparatus for detecting and/or discriminating an object. The detectingapparatus is capable of detecting weak light emitted from the object.

Hereinafter, embodiments consistent with the present invention will bedescribed in detail with reference to drawings. Wherever possible, thesame reference numbers will be used throughout the drawings to refer tothe same or like parts.

1. Apparatus of the Invention

The detecting apparatus consistent with the present invention can beused to detect and/or discriminate an object, which is capable ofemitting light. The object may be a source of luminescence, such as afluorescent dye molecule, a phosphorescent dye molecule, a quantum dot,or a luminescent product (e.g., an excited-state reaction product suchas singlet oxygen or excited coelenteramide) of a bioluminescent orchemiluminescent system. An apparatus of the invention may or may notcomprise at least one source of excitatory light, such as at least onelaser. A source of excitatory light is not needed to detect objectswhich luminesce independently of light absorption, such as can begenerated via bioluminescence or chemiluminescence, for example. Theobject may also be a target molecule without light emitting capability,but may be attached to a labeling object which is capable of emittinglight (e.g., a fluorescent dye molecule, a phosphorescent dye molecule,or a quantum dot). A certain labeling object may be capable of beingattached to a specific target molecule. Thus, the target molecule may beidentified via the labeling object. More than one labeling object may beattached to one target molecule. The apparatus may also be used tomonitor a large number of objects.

The detecting apparatus consistent with the present invention maycomprise a light detector detecting light emitted from the object. Thelight detector is capable of at least partially absorbing light incidentthereon and generating output signals in response to the light. Thelight detector may comprise a control circuit for controlling theoperation of the light detector. The control circuit may comprise acircuit of signal amplifier, A/D convertor, integrator, comparator,logic circuit, readout circuit, memory, microprocessor, clock, and/oraddress.

The detecting apparatus may comprise a computer for processing outputsignals from the light detector and generating a determination result.The detecting apparatus may further comprise a blind sheet with apinhole. The detecting apparatus may also comprise a linker site towhich the object may be attached. The linker site may be formed in thepinhole or be formed outside but proximate to the pinhole. The apparatusmay further comprise an excitation light source. The object may absorblight emitted from the excitation light source and then emit anotherlight to be detected by the detecting apparatus. The light emitted fromthe object may have different wavelength than the light emitted from theexcitation light source.

1.1 Optical Sensor

The light detector consistent with the present invention may comprise aplurality of optical sensors. An optical sensor consistent with thepresent invention may be, e.g., a photodiode, an avalanche photodiode(APD), a phototransistor, a photogate, a quantum-well infraredphotodetector (QWIP), a thin-film on ASIC (TFA), ametal-semiconductor-metal (MSM) photodetector, or a combination thereof.In one embodiment of the present invention, the optical sensor may be aphotodiode.

A photodiode is a solid-state device which converts light into electriccurrent or voltage. A photodiode is usually made of semiconductormaterials, such as silicon. The basic structure of a photodiodecomprises a p-n junction formed by joining a p-type semiconductor and ann-type semiconductor. A photodiode may have only one p-n junction andprovide only one output signal, therefore be considered as one opticalsensor; a multi-junction photodiode has more than one p-n junction andmay provide more than one output signal, wherein each p-n junction maybe considered as one optical sensor.

Alternatively, a photodiode may be a p-i-n photodiode. The p-i-nphotodiode is a special type of p-n junction photodiode with anintrinsic layer, or a lightly-doped n-type layer, or a lightly-dopedp-type layer, sandwiched between the n-type layer and the p-type layer.In a p-i-n photodiode, the thickness of the depletion region, which isalmost the whole intrinsic layer, can be tailored to optimize thequantum efficiency and frequency response. (S. M. Sze, Physics ofSemiconductor Devices, pp. 674-675, Wiley, 2007).

P-type semiconductor and n-type semiconductor may be formed by dopingp-type impurities and n-type impurities into a semiconductor substrate,respectively. For example, in silicon, phosphorus or arsenic may act asn-type impurities, and boron and indium may act as p-type impurities.Generally, two methods may be used to introduce impurities into asemiconductor substrate: diffusion and ion implantation. By diffusing orimplanting p-type impurities into an n-type semiconductor substrate,portion of the n-type semiconductor substrate may be converted to p-typesemiconductor. Accordingly, a p-n junction may be formed. Alternatively,by diffusing or implanting n-type impurities into an p-typesemiconductor substrate, portion of the p-type semiconductor substratemay be converted to n-type semiconductor, and thus a p-n junction may beformed. Other than diffusion and ion implantation, epitaxial growth mayalso be used to fabricate n-type semiconductor or p-type semiconductor,which is a process that a thin, single crystal layer is grown on thesurface of a single crystal substrate. (D. A. Neamen, SemiconductorPhysics & Devices, pp. 17-18, McGrawHill, 1997). When a light isincident on a photodiode, electron-hole pairs may be generated in thephotodiode. If an electron-hole pair is generated in the depletionregion of the photodiode, the built-in potential in the depletion regionmay separate the electron and the hole and thus generate eitherphotocurrent or voltage.

Different photodiodes may have different capabilities of absorbing anincident light. The capability can be described by the absorptioncoefficient α of the material constituting the photodiode. FIG. 1 showsthe absorption coefficient of silicon at different wavelengths. Thephoton flux φ of the incident light decreases exponentially with thedepth that the light penetrates into the photodiode. The photon flux ata depth x inside the photodiode can be expressed as:φ(λ,x)=φ₀(λ)−e ^(−αx)where λ is the wavelength of the incident light and φ₀ is the photonflux at the surface of the photodiode. Therefore, the photon fluxabsorbed in the depletion region can be given by:Δφ(λ)=φ₀(λ)·(e ^(−αxu) −e ^(−αxl))where xu and xl are the depths of the upper and lower boundaries of thedepletion region, respectively.

The photon flux absorbed in the depletion region may be converted to aphotocurrent as an output signal of the photodiode. The photocurrent canbe represented by:I _(PD)(λ)=1.98×10⁻¹⁶ ·A·[φ₀(λ)·R(λ)/λ]where A is the area of the optical window, and R(λ) is the responsivityof a photodiode, which is defined as the amount of photocurrent producedby a unit power of incident light.1.2 Computer for Signal Processing

Consistent with the present invention, a computer is provided which isable to process output signals generated by the light detector byperforming a mathematic or logic operation on the output signals, and todetermine the presence and type of an object. The computer used forprocessing output signals from an optical sensor may be, for example, apersonal computer, a microprocessor-based or programmable consumerelectronics, a logic circuit, or the like.

The computer may comprise one or more storage devices that temporarilyor permanently store data and computer program containing software codesrepresenting an algorithm. The computer may perform processing based onthe algorithm. The storage device may be a read-only memory (ROM), arandom access memory (RAM), or a memory with other access options. Thestorage device may be physically implemented by computer-readable media,such as, for example: (a) magnetic media, like a hard disk, a floppydisk, or other magnetic disk, a tape, a cassette tape; (b) opticalmedia, like optical disk (CD-ROM, digital versatile disk—DVD); (c)semiconductor media, like DRAM, SRAM, EPROM, EEPROM, memory stick, or byany other media, like paper.

The computer may comprise one or more interfaces for receiving outputsignals from the light detector. The computer may comprise one or moreprocessors for processing these signals.

Moreover, the computer may comprise one or more input devices forreceiving commands from a user, and may comprise one or more outputdevices for outputting the processing results. The input device may be,for example, a mouse, a keyboard, or a control panel having a pluralityof buttons. The output device may be, for example, a display or aprinter.

1.3 Exemplary Detecting Apparatuses

Referring to FIG. 2, a detecting apparatus 1 consistent with the presentinvention is illustrated. The detecting apparatus 1 may comprise a lightdetector 11 and a computer 12.

The light detector 11 may comprise a first optical sensor 111 and asecond optical sensor 112. In some embodiments, the light detector 11may comprise more than two optical sensors. For example, in someembodiments, the light detector 11 may comprise three optical sensors orfour optical sensors. The first optical sensor 111 and the secondoptical sensor 112 may be stacked. In the stacked configuration, lightemitted from an object passes through the first optical sensor 111, andthen passes through the second optical sensor 112. In some embodiments,the optical sensors may be vertically or substantially verticallystacked. In other embodiments, the optical sensors may be obliquelystacked in a direction at a certain angle (such as 10°, 30°, 45°, 60°,etc.) with respect to the vertical direction. In some embodiments, thelight detector 11 may be a multi-junction photodiode comprising aplurality of p-n junctions formed therein. In a multi-junctionphotodiode, each p-n junction may form the basic structure of oneindividual optical sensor. Each of the first optical sensor 111 and thesecond optical sensor 112 may comprise, for example, one p-n junction inthe multi-junction photodiode, respectively, as shown in FIG. 3.

In some embodiments, different optical sensors in the light detector 11may be made of same semiconductor material, such as silicon, germanium,GaAs AlGaAs, InGaAs, InGaAsN, InGaP, CdTe, or CdS. In some embodiments,different optical sensors in the light detector 11 may be made ofdifferent semiconductor materials. For example, FIG. 4 shows a lightdetector formed of two vertically stacked optical sensors PD-1 and PD-2.Optical sensors PD-1 is made of material-1 and material-2 while PD-2 ismade of material-2 and material-3. Material-1, material-2, andmaterial-3 may be a combination of the semiconductor materials mentionedabove. In addition, the thickness of these optical sensors may also bedifferent.

As shown in FIG. 2, light emitted from an object, e.g., a quantum dot(QD) incident on the light detector 11 may be partially absorbed by thefirst optical sensor 111, generating a first signal J1. Unabsorbed lightpasses through the first optical sensor 111 and may then be partiallyabsorbed by the second optical sensor 112, generating a second signalJ2. These signals may be sent to the computer 12 for processing.

In some embodiments, light emitted from an object may be incident on thelight detector 11 without passing through another optical element, suchas an optical filter, a prism, or a lens (i.e., may be directly incidenton the light detector). In some embodiments, the apparatus may furthercomprise a light collecting optical structure for collecting the lightemitted from the object.

In some embodiments, the light detector 11 may comprise two or moreoptical sensors arranged horizontally (not shown). In these embodiments,the detecting apparatus 1 may further comprise an optical structureplaced between the object and the light detector 11. The light emittedfrom the object may be directed by the optical structure to one or moreof the optical sensors and partially absorbed by the one or more of theoptical sensors. The direction of light after passing through theoptical structure may be different for light with different wavelength.Therefore, different optical sensors in the light detector withhorizontal arrangement may detect light with different wavelengths.

In some embodiments, the optical structure may comprise a prism. In someembodiments, the optical structure may comprise a lens. In someembodiments, the optical structure may comprise a prism and a lens. Theprism may be, e.g., a triangular prism, an Abbe prism, a Pellin-Brocaprism, an Amici prism, or a combination thereof. The lens may be, e.g.,a converging lens, a diverging lens, or a combination thereof. Theoptical structure may be made from, e.g., glass, plastic, or othermaterials that are transparent to the wavelengths of interest. Thetransparency of the optical structure may be high enough to allow themajority of the light to pass through the optical structure.

Alternatively, in some embodiments, the optical structure may comprise agrating. The grating may be, e.g., a binary grating, a blaze grating, asinusoidal grating, a multi-level grating, a volume grating, or acombination thereof. FIG. 5 shows as an example a detecting apparatusconsistent with one embodiment of the present invention. In thisdetecting apparatus, the light detector comprises a plurality of opticalsensors horizontally arranged at different positions. A grating may beused to direct light of different wavelengths from an object todifferent positions of the light detector so as to be absorbed bydifferent optical sensors.

Consistent with the present invention, a detecting system may beprovided comprising an array of the detecting apparatuses consistentwith the present invention. Consistent with the present invention, adetecting system may also be provided comprising an array of the lightdetectors consistent with the present invention and a computer forprocessing output signals from the array of light detectors andperforming detection and/or discrimination based on the processingresult. Such detecting systems may detect and/or discriminate aplurality of objects at one time, and thus may be suitable forlarge-scale parallel sequencing of nucleic acids, for example.

The computer 12 may comprise a processing unit 121 and an output unit122. The processing unit 121 processes the output signals from the lightdetector 11 and determines the presence and type of an object. Outputunit 122 outputs the determination result. In some embodiments, thecomputer 12 may also comprise a storage device (not shown), storing acomputer program. The computer program may contain software codes forinstructing the computer to process the signals.

The light detector 11 and computer 12 may be integrated into a singlepiece of semiconductor chip. The light detector 11 and computer 12 maybe formed on separated semiconductor chips.

2. Methods of Detection

Light emitted from different objects may have different spectra. Forexample, FIG. 6 shows the emission spectra of two quantum dots (“QDs”)(QD-1 and QD-2) as two objects. In this example, the peaks of these twoemission spectra are at different wavelengths. In other examples, theemission spectra of different objects may also have different shapes.

The first optical sensor 111 and the second optical sensor 112 may havedifferent responsive properties. FIG. 7 shows, as an example, theresponsivity curves of the first optical sensor 111 and the secondoptical sensor 112. The responsivities of the first optical sensor 111and the second optical sensor 112 may differ from each other in thatthey have different shapes and/or they may peak at differentwavelengths.

For clearer illustration, FIG. 8 shows the responsivity curves of twooptical sensors and the emission spectra of two QDs in one figure. Inthis example, light emitted from QD-1 may generate signals of similarstrength at the two optical sensors. On the other hand, light emittedfrom QD-2 may generate a signal at the second optical sensor 112stronger than that at the first optical sensor 111.

In some embodiments, as shown in FIG. 2, light emitted from an objectmay be incident on the light detector 11 and partially absorbed by thefirst optical sensor 111 and the second optical sensor 112, which inturn generate signals J1 and J2, respectively. The computer 12 processesthe signals J1 and J2, and discriminates the object.

FIG. 9 shows a flow chart of a method for discriminating an objectaccording to one embodiment of the present invention. At step 201, alight emitted from an object is incident on the light detector 11. Atstep 202, two output signals J1 and J2 are generated by the firstoptical sensor and the second optical sensor, respectively, and thesignals J1 and J2 are sent to the computer 12. At step 203, the computer12 processes the signals J1 and J2. At step 204, the computer 12compares the processing result with a known result corresponding to aknown type of object. At step 205, the computer 12 determines whetherthe object belongs to the known type.

In one embodiment, processing the signals J1 and J2 may comprisecalculating a ratio of J1 and J2. If the calculated ratio is within acertain range corresponding to the known type of object, it may bedetermined that the object being detected belongs to the known type. Forexample, a known type of quantum dot QD-1 may have a corresponding rangeof 0.7<J2/J1<1.2. If the calculated ratio for an object being detectedis 0.9, it may be determined that the object is a QD-1 type object.

FIG. 10 shows a flow chart of a method for discriminating an objectaccording to another embodiment of the present invention. In thisembodiment, an object may be determined to belong to one of, e.g., threeknown types of object, i.e., Type 1, Type 2, and Type 3. The first twosteps of this embodiment may be the same as those in the above-notedembodiment. At step 303, the computer 12 determines the relationshipbetween these two signals J1 and J2. At step 304, the computer 12compares the relationship with known results corresponding to knowntypes of object. At step 305, the computer 12 determines to which typethe object belongs based on the comparison result.

In this embodiment, for example, if J1>J2, then the object may bedetermined to be a Type 1 object. If J1≈J2, then the object may bedetermined to be a Type 2 object. If J1<J2, then the object may bedetermined to be a Type 3 object.

FIG. 11 shows a flow chart of a method for discriminating an objectaccording to still another embodiment of the present invention. In thisembodiment, an object may be determined to belong to one of a pluralityof known types of object, i.e., Type 1 to Type n. The first two steps ofthis embodiment may be the same as those in the above-noted embodiment.At step 403, the computer calculates the ratio of J1 and J2. At step404, the computer 12 compares the calculated ratio with known ratioscorresponding to known types of object. At step 405, the computer 12determines to which type the object belongs based on the comparisonresult.

In this embodiment, for example, if, among all the known ratios of theknown types, the known ratio of Type i (1≤i≤n) is closest to thecalculated ratio, then the object may be determined to be a Type iobject.

FIG. 12 shows a flow chart of a method for discriminating an objectaccording to a further embodiment of the present invention. In thisembodiment, an object may be determined to belong to one of a pluralityof known types of object, i.e., Type 1 to Type n. The first three stepsof this embodiment may be the same as those in the above-notedembodiment. At step 504, the computer 12 compares the calculated ratiowith known ratio ranges corresponding to known types of object. At step505, the computer 12 determines to which type the object belongs basedon the comparison result.

In this embodiment, for example, if the calculated ratio falls withinthe ratio range corresponding to Type i (1≤i≤n), then the object may bedetermined to be a Type i object. If the calculated ratio does not fallwithin any of the ranges, it may be reported that the confidence levelis low and that the type of the object being detected may not bedetermined. For example, assuming Type 1 object has a correspondingratio range of 0.7<J2/J1<1.2 and Type 2 object has a corresponding ratiorange of J2/J1>2, if the calculated ratio for an object being detectedis 1, then it may be determined that the object is a Type 1 object. Onthe other hand, if the calculated ratio for an object being detected is1.5, then it may be reported that the confidence level is low and thatthe type of the object may not be determined.

In one embodiment, the ratio of J1 and J2 may be J2/J1. In oneembodiment, the ratio of J1 and J2 may be J1/J2. In some embodiments,the ratio of J1 and J2 may be J2/(c×J1) or J1/(c×J2), where “c” is acoefficient.

In some embodiments, after signals are sent to the computer 12, thecomputer 12 may additionally perform a step of determining whether anobject is present in a sample. In some embodiments, the determinationmay be performed by comparing the sum of all or some of the signals witha threshold value. In some embodiments, for example, if the sum is equalto or larger than the threshold, it may be determined that an object ispresent. On the other hand, if the sum is smaller than the threshold, itmay be determined that an object is absent. In other embodiments, if thesum is smaller than the threshold value, it may be determined that anobject is present. If the sum is equal to or larger than the thresholdvalue, it may be determined that an object is absent. In someembodiments, the determination may be performed by comparing the signalsdirectly with a threshold to determine the presence of an object. Forexample, if any signal is, or a certain number of signals are, equal toor larger than the threshold, it may be determined that an object ispresent. On the other hand, if all the signals are smaller than thethreshold, it may be determined that an object is absent.

In some embodiments, the light detector 11 may comprise three or moreoptical sensors, each generates an output signal upon the light emittedfrom an object incident on the light detector 11. In some embodiments,two of the generated output signals may be processed by the computer 12to discriminate the object. In other embodiments, more or all of thegenerated output signals may be processed by the computer 12 todiscriminate the object.

FIG. 13 shows a flow chart of a method for discriminating an objectaccording to yet another embodiment of the present invention. In thisembodiment, the light detector 11 comprises three optical sensors PD1,PD2, and PD3. An object in a sample may be detected and determined tobelong to, e.g., one of four types, such as dye 1, dye 2, dye 3, and dye4. Output signals generated by these three optical sensors arerepresented by I_(PD1), I_(PD2), and I_(PD3), respectively, and sent tothe computer 12 for processing. The method is described below.

First, the sample may be excited using an excitation light source.Optical sensors PD1, PD2, and PD3 may then generate output signalsI_(PD1), I_(PD2), and I_(PD3) Upon receiving the output signals, thecomputer 12 may calculate a sum of these signals. If the sum is smallerthan a threshold value V_(th), the computer 12 may generate a reportthat no fluorescence occurred. Otherwise, the computer may proceed tothe next step.

When it is determined that fluorescence did occur and that an objectexists, the computer 12 may first perform an original discriminationstep. In this step, the computer 12 may calculate a first ratio betweenI_(PD3) and the sum of all signals, and compare this ratio with a firstset of threshold values, e.g., V_(th1), V_(th2), V_(th3), and V_(th4),to determine whether the calculated first ratio falls within any rangebetween 0 and V_(th1), between V_(th1) and V_(th2), between V_(th2) andV_(th3), or between V_(th3) and V_(th4). If, however, the calculatedfirst ratio does not fall in any of the above ranges, the computer 12may generate a report that a fault emission occurred.

After that, a confirmation step is performed. In the confirmation step,a second ratio between I_(PD2) and I_(PD3) is calculated, and iscompared with a second set of threshold values, e.g., V_(th1)′,V_(th2)′, V_(th3)′, and V_(th4)′. to determine whether the calculatedsecond ratio falls within any range between 0 and V_(th1)′, betweenV_(th1)′ and V_(th2)′, between V_(th2)′ and V_(th3)′, or betweenV_(th3)′ and V_(th4)′.

For example, if, in the original discrimination step, the calculatedfirst ratio falls within the range between V_(th1) and V_(th2), it maybe determined that the object possibly belongs to the type dye 2. Thecomputer 12 may then proceed to the confirmation step to determinewhether the calculated second ratio falls within the range betweenV_(th1)′ and V_(th2)′. If yes, it may be determined that the objectbelongs to the type dye 2. Otherwise, the computer 12 may generate areport that a fault emission occurred.

It is noted that any one of the original discrimination step and theconfirmation step described above may be performed individually todiscriminate an object. Performing both two steps in one discriminationprocedure, however, may improve the accuracy.

FIG. 14 shows a flow chart of a method for discriminating an objectaccording to yet a further embodiment of the present invention. In thisembodiment, the light detector 11 comprises a plurality of opticalsensors PD1, PD2, . . . PDn, where n is larger than 1 and may be equalto or smaller than 6. Moreover, n may be equal to 3. The apparatus andmethod of this embodiment may be used to detect and discriminate qdifferent objects, e.g., q different dyes, denoted as D1, D2, . . . Dq.

The excitation light source used in this embodiment may have k differentexcitation light bands, L1, L2, . . . Lk, where k may be equal to orlarger than 1 and equal to or smaller than 3. The excitation lightsource may turn on and off following a set of instructions, resulting ina set of m different excitation conditions Cj, where 1≤j≤m. Eachexcitation condition may be a combination of different excitation lightbands. Under the same excitation condition, the output signal generatedby a certain optical sensor for different dyes may be different.

The output signal generated at the i-th optical sensor PDi (where 1≤i≤n)under the j-th excitation condition Cj for a certain dye Ds (where1≤s≤q) may be denoted as Ds{J{PDi:Cj}}. Different combinations of PDiand Cj may result in different Ds{J{PDi:Cj}}. Therefore, before usingthe apparatus to detect and discriminate an unknown dye, by performingcalibration reading using different combinations of PDi and Cj atstandard environments for a dye Ds, one may obtain a matrix of outputsignals. This matrix may be unique to a dye, and may be called astandard reading matrix for a dye Ds, M{dye Ds}, as shown below:

$\begin{matrix}{{{Ds}\left\{ {J\left\{ {{{PD}\; 1}:{C\; 1}} \right\}} \right\}},} & {{{Ds}\left\{ {J\left\{ {{{PD}\; 2}:{C\; 1}} \right\}} \right\}},} & \ldots & {{Ds}\left\{ {J\left\{ {{PD}\;{n:{C\; 1}}} \right\}} \right\}} \\{{{Ds}\left\{ {J\left\{ {{{PD}\; 1}:{C\; 2}} \right\}} \right\}},} & {{{Ds}\left\{ {J\left\{ {{{PD}\; 2}:{C\; 2}} \right\}} \right\}},} & \ldots & {{Ds}\left\{ {J\left\{ {{PD}\;{n:{C\; 2}}} \right\}} \right\}} \\\ldots & \; & \; & \; \\{{{Ds}\left\{ {J\left\{ {{{PD}\; 1}:{C\; m}} \right\}} \right\}},} & {{{Ds}\left\{ {J\left\{ {{{PD}\; 2}:{C\; m}} \right\}} \right\}},} & \ldots & {{Ds}\left\{ {J\left\{ {{PD}\;{n:{C\; m}}} \right\}} \right\}}\end{matrix}\quad$This calibration reading may be repeated for each of the dyes, creatingq standard reading matrices M{dye D1}, M{dye D2}, . . . M{dye Dq}.

To minimize the impact of the background, a background reading may alsobe obtained by recording output signals from the optical sensors when nodye exists, which may result in a background reading matrixM{background}:

$\begin{matrix}{{B\left\{ {J\left\{ {{{PD}\; 1}:{C\; 1}} \right\}} \right\}},} & {{B\left\{ {J\left\{ {{{PD}\; 2}:{C\; 1}} \right\}} \right\}},} & \ldots & {B\left\{ {J\left\{ {{PD}\;{n:{C\; 1}}} \right\}} \right\}} \\{{B\left\{ {J\left\{ {{{PD}\; 1}:{C\; 2}} \right\}} \right\}},} & {{B\left\{ {J\left\{ {{{PD}\; 2}:{C\; 2}} \right\}} \right\}},} & \ldots & {B\left\{ {J\left\{ {{PD}\;{n:{C\; 2}}} \right\}} \right\}} \\\ldots & \; & \; & \; \\{{B\left\{ {J\left\{ {{{PD}\; 1}:{C\; m}} \right\}} \right\}},} & {{B\left\{ {J\left\{ {{{PD}\; 2}:{C\; m}} \right\}} \right\}},} & \ldots & {B\left\{ {J\left\{ {{PD}\;{n:{C\; m}}} \right\}} \right\}}\end{matrix}\quad$

When an unknown sample is applied to the detecting apparatus, outputsignals generated by the optical sensors at different excitationconditions are measured and a sample reading matrix M{sample} isobtained:

$\begin{matrix}{{J\left\{ {{{PD}\; 1}:{C\; 1}} \right\}},} & {{J\left\{ {{{PD}\; 2}:{C\; 1}} \right\}},} & \ldots & {J\left\{ {{PD}\;{n:{C\; 1}}} \right\}} \\{{J\left\{ {{{PD}\; 1}:{C\; 2}} \right\}},} & {{J\left\{ {{{PD}\; 2}:{C\; 2}} \right\}},} & \ldots & {J\left\{ {{PD}\;{n:{C\; 2}}} \right\}} \\\ldots & \; & \; & \; \\{{J\left\{ {{{PD}\; 1}:{C\; m}} \right\}},} & {{J\left\{ {{{PD}\; 2}:{C\; m}} \right\}},} & \ldots & {J\left\{ {{PD}\;{n:{C\; m}}} \right\}}\end{matrix}\quad$

This sample reading matrix may then be compared with the standardreading matrices to determine which type of dye the sample contains. Tominimize the impact of the background on the result, the backgroundreading matrix may be subtracted from both the standard reading matricesand the sample reading matrix before comparison.

The comparison may be carried out by calculating a rank Rs for each typeof dye Ds. When calculating Rs, weight factors and statistic methods maybe applied. For example, a least squared method or a most likelihoodmethod may be used to find the best match. Weight factors may be appliedto each optical sensor or excitation light mode to increase the accuracyof the analysis.

After Rs is calculated, the computer 12 may report the most probabledye, the second probable dye, and so on according to the rank Rs.

In the Sequencing by Synthesis method, DNA sequence is determined byidentifying the newly added nucleotide to the growing strand. Thenucleotide adding process is detected by fluorescent light emitted fromthe fluorophore attached to the nucleotide. The nucleotide incorporationreaction can be divided into several steps: the nucleotide docking intoactive site formed by polymerase and template, breaking the phosphatebond, and forming the new bond to the sugar. Some nucleotides justdiffuse in and out the active site, with no real incorporationhappening. To monitor the incorporation reaction in real time, thedetecting apparatus may need to be able to detect a fluorophore coming,time of retention, and type of fluorophore. The reason of measuringretention time is that the diffusing nucleotides may stay for a shorterperiod of time than those incorporated nucleotides. By setting aretention time threshold, a real incorporation event can be detected. Aflow chart of a method according to still a further embodiment of thepresent invention, which may be used for event detection is shown inFIG. 15. This method is basically similar to that described above, withone more step added to determine whether an event can be claimed.Therefore, detailed description of this method is omitted. FIG. 16 showsa block diagram of one example detecting apparatus which may be used inthis embodiment.

In some embodiments, the light detector 11 may comprise two or moreoptical sensors arranged horizontally. The signals generated by thehorizontally arranged optical sensors may be input to the computer 12and processed in a manner similar to one of the methods disclosed aboveto determine the presence of an object and/or to discriminate the typeof an object.

3. Applications

The detecting apparatuses and systems consistent with the presentinvention, and method of using the same may be applied to, e.g., nucleicacid detection, DNA sequencing, biomarker identification, or flowcytometry. The detecting apparatuses can detect and process lowintensity light signal, which makes single molecule objectdiscrimination possible.

In some embodiments of the methods of the present invention, labels areattached to the analyte(s) (i.e., the substance(s) to be detected), theprobe(s), such as primers, antibodies, or other reagents that interactwith the analyte(s), or other reagent(s), such as nucleotides (includingnucleotide analogs). Any label can be used on the analyte or probe whichcan be useful in the correlation of signal with the amount or presenceof analyte.

For example, a wide variety of fluorescent molecules can be utilized inthe present invention including small molecules, fluorescent proteinsand quantum dots. Useful fluorescent molecules (fluorophores) include,but are not limited to: 1,5 IAEDANS; 1,8-ANS; 4-Methylumbelliferone;5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5-FAM);5-Carboxynapthofluorescein; 5-Carboxytetramethylrhodamine (5-TAMRA);5-FAM (5-Carboxyfluorescein); 5-HAT (Hydroxy Tryptamine); 5-HydroxyTryptamine (HAT); 5-ROX (carboxy-X-rhodamine); 5-TAMRA(5-Carboxytetramethylrhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G; 6-JOE;7-Amino-4-methylcoumarin; 7-Aminoactinomycin D (7-AAD);7-Hydroxy-4-methylcoumarin; 9-Amino-6-chloro-2-methoxyacridine; ABQ;Acid Fuchsin; ACMA (9-Amino-6-chloro-2-methoxyacridine); AcridineOrange; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin FeulgenSITSA; AFPs-AutoFluorescent Protein-(Quantum Biotechnologies); TexasRed; Texas Red-X conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R;Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TCN; Thiolyte;Thiozole Orange; Tinopol CBS (Calcofluor White); TMR; TO-PRO-1;TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITC(TetramethylRodaminelsoThioCyanate); True Blue; TruRed; Ultralite;Uranine B; Uvitex SFC; WW 781; X-Rhodamine; XRITC; Xylene Orange; Y66F;Y66H; Y66W; YO-PRO-1; YO-PRO-3; YOYO-1; interchelating dyes such asYOYO-3, Sybr Green, Thiazole orange; members of the Alexa Fluor dyeseries (from Molecular Probes/Invitrogen) which cover a broad spectrumand match the principal output wavelengths of common excitation sourcessuch as Alexa Fluor 350, Alexa Fluor 405, 430, 488, 500, 514, 532, 546,555, 568, 594, 610, 633, 635, 647, 660, 680, 700, and 750; members ofthe Cy Dye fluorophore series (GE Healthcare), also covering a widespectrum such as Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7; members of theOyster dye fluorophores (Denovo Biolabels) such as Oyster-500, -550,-556, 645, 650, 656; members of the DY-Labels series (Dyomics), forexample, with maxima of absorption that range from 418 nm (DY-415) to844 nm (DY-831) such as DY-415, -495, -505, -547, -548, -549, -550,-554, -555, -556, -560, -590, -610, -615, -630, -631, -632, -633, -634,-635, -636, -647, -648, -649, -650, -651, -652, -675, -676, -677, -680,-681, -682, -700, -701, -730, -731, -732, -734, -750, -751, -752, -776,-780, -781, -782, -831, -480XL, -481XL, -485XL, -510XL, -520XL, -521XL;members of the ATTO series of fluorescent labels (ATTO-TEC GmbH) such asATTO 390, 425, 465, 488, 495, 520, 532, 550, 565, 590, 594, 610, 611X,620, 633, 635, 637, 647, 647N, 655, 680, 700, 725, 740; members of theCAL Fluor series or Quasar series of dyes (Biosearch Technologies) suchas CAL Fluor Gold 540, CAL Fluor Orange 560, Quasar 570, CAL Fluor Red590, CAL Fluor Red 610, CAL Fluor Red 635, Quasar 670; quantum dots,such as quantum dots of the EviTags series (Evident Technologies) orquantum dots of the Qdot series (Invitrogen) such as the Qdot 525,Qdot565, Qdot585, Qdot605, Qdot655, Qdot705, Qdot 800; fluorescein;rhodamine; and/or phycoerythrin; or combinations thereof. See, e.g.,U.S. Application Publication 2008/0081769.

In some embodiments, at least one bioluminescent or chemiluminescentsystem is provided which generates light in the presence of an entitysuch as an analyte, reagent, or reaction product. For example, abioluminescent or chemiluminescent system can be used to detectpyrophosphate generated in a sequencing by synthesis reaction (discussedin more detail below); to detect the presence of metals such as iron orcopper by their catalysis of a light-generating reaction; or to measurethe amount of a reagent bound by an analyte, wherein the reagentcomprises at least one component of the bio- or chemi-luminescentsystem.

Examples of bioluminescent systems known in the art include systemscomprising at least one luciferase, e.g., firefly luciferases, includingPhotinus pyralis luciferase. A bioluminescent system can be used todetect pyrophosphate, for example, by providing luciferase, ATPsulfurylase, luciferin, and adenosine 5′ phosphosulfate, together withthe components of the sequencing by synthesis reaction (in which dATPcan be substituted with an analog such as dATPαS to avoid nonspecificlight due to consumption of dATP by luciferase). When pyrophosphate isgenerated by a nucleotide incorporation event, ATP sulfurylase producesATP in an adenosine 5′ phosphosulfate dependent manner. The ATP drivesconversion of luciferin to oxyluciferin plus light by luciferase. Otherbioluminescent systems include systems based on photoproteins such asaequorin, which oxidizes coelenterazine to excited coelenteramide, whichemits light.

Examples of chemiluminescent systems include luminol plus hydrogenperoxide, which can undergo a light-emitting reaction in the presence ofa metal catalyst or auxiliary oxidant; diphenyl oxalate plus hydrogenperoxide and a suitable dye, which undergoes excitation and lightemission in a multistep reaction that produces carbon dioxide (examplesof suitable dyes include phenylated anthracene derivatives such as9,10-diphenylanthracene, 9,10-Bis(phenylethynyl)anthracene, and1-Chloro-9,10-bis(phenylethynyl)anthracene, and rhodamines such asrhodamine 6G and rhodamine B); singlet oxygen-producing systems such ashydrogen peroxide plus sodium hypochlorite; and systems comprising anenzyme such as horseradish peroxidase, which acts on luminol or othercommercially available substrates.

In some embodiments, the methods of the invention comprise formingcovalent attachments, such as between reagents or analytes and surfacesor labels. For example, in single molecule sequencing procedures, anucleic acid molecule or an enzyme such as a polymerase may be attachedto a surface such as a glass slide. Such an attachment can allow theacquisition of data over multiple sequencing cycles. In someembodiments, Many methods for forming covalent attachments, such as ofreagents to surfaces or labels, are known in the art. Non-covalentattachment methods can also be used. A number of different chemicalmodifiers can be used to facilitate attachment formation. Examples ofchemical modifiers include N-hydroxy succinimide (NHS) groups, amines,aldehydes, epoxides, carboxyl groups, hydroxyl groups, hydrazides,hydrophobic groups, membranes, maleimides, biotin, streptavidin, thiolgroups, nickel chelates, photoreactive groups, boron groups, thioesters,cysteines, disulfide groups, alkyl and acyl halide groups, glutathiones,maltoses, azides, phosphates, and phosphines. Surfaces such as glassslides with such chemically modified surfaces are commercially availablefor a number of modifications. These can easily be prepared for therest, using standard methods (Microarray Biochip Technologies, MarkSchena, Editor, March 2000, Biotechniques Books). In some embodiments,attachments are formed between two entities by using an appropriatecombination of modifiers (e.g., an electrophilic modifier and anucleophilic modifier), wherein each entity comprises at least onemodifier.

In some embodiments, attachments are formed between two entities byusing a chemical modifier present on one of the entities and a naturallyoccurring moiety, for example, an amine or sulfhydryl, of the otherentity. In some embodiments, modifiers that are reactive to amines areused. An advantage of this reaction is that it can be fast and can avoidproduction of toxic by-products. Examples of such modifiers includeNHS-esters, aldehydes, epoxides, acyl halides, and thio-esters. Mostproteins, peptides, glycopeptides, etc., have free amine groups, whichcan react with such modifiers to link them covalently to thesemodifiers. Nucleic acid probes with internal or terminal amine groupscan also be synthesized, and are commercially available (e.g., from IDTor Operon). Thus, biomolecules can be bound (e.g., covalently ornon-covalently) to labels, surfaces, or other reagents using similarchemistries.

A number of other multi-functional cross-linking agents can be used toconvert the chemical reactivity of one kind of modifier to another.These groups can be bifunctional, tri-functional, tetra-functional, andso on. They can also be homo-functional or hetero-functional. An exampleof a bi-functional cross-linker is X-Y-Z, where X and Z are two reactivegroups, and Y is a connecting linker. Further, if X and Z are the samegroup, such as NHS-esters, the resulting cross-linker, NHS-Y-NHS, is ahomo-bi-functional cross-linker and could connect two entities that eachcomprise an amine. If X is NHS-ester and Z is a maleimide group, theresulting cross-linker, NHS-Y-maleimide, is a hetero-bi-functionalcross-linker and could link an entity comprising an amine with an entitycomprising a thio-group. Cross-linkers with a number of differentfunctional groups are widely available. Examples of such functionalgroups include NHS-esters, thio-esters, alkyl halides, acyl halides(e.g., iodoacetamide), thiols, amines, cysteines, histidines,di-sulfides, maleimide, cis-diols, boronic acid, hydroxamic acid,azides, hydrazines, phosphines, photoreactive groups (e.g.,anthraquinone, benzophenone), acrylamide (e.g., acrydite), affinitygroups (e.g., biotin, streptavidin, maltose, maltose binding protein,glutathione, glutathione-S-transferase), aldehydes, ketones, carboxylicacids, phosphates, hydrophobic groups (e.g., phenyl, cholesterol), etc.

Other modifier alternatives (such as photo-crosslinking and thermalcrosslinking) are known to those skilled in the art. Commerciallyavailable technologies include, for example, those from MosiacTechnologies (Waltham, Mass.), EXIQON™ (Vedbaek, Denmark), Schleicherand Schuell (Keene, N.H.), Surmodics™ (St. Paul, Minn.), XENOPORE™(Hawthorne, N.J.), Pamgene (Netherlands), Eppendorf (Germany), Prolinx(Bothell, Wash.), Spectral Genomics (Houston, Tex.), and COMBIMATRIX™(Bothell, Wash.).

In some embodiments, surfaces other than glass are provided. Forexample, metallic surfaces, such as gold, silicon, copper, titanium, andaluminum, metal oxides, such as silicon oxide, titanium oxide, and ironoxide, and plastics, such as polystyrene, and polyethylene, zeolites,and other materials can also be used. In some embodiments, the layers ofthese materials can be thin, e.g., less than about 100 nm in order toallow the transmission of light.

3.1 Nucleic Acid Detection

A detecting apparatus consistent with the present invention may be usedas part of a system for or in methods or processes of moleculedetection, e.g., nucleic acid sequencing. This apparatus, and methods orprocesses utilizing it, are useful for, e.g., analytical and diagnosticapplications. These applications may be private, public, commercial, orindustrial.

A detecting apparatus consistent with the present invention may be usedwith a wide variety of sequencing modalities and may be suitable forsequencing single molecules. Additionally, the detecting apparatusconsistent with the present invention have simplified design, assembly,and production relative to existing biochip devices. For example, thenucleic acids to be sequenced may be affixed to random linker sites onthe array of the system, avoiding the use of time consuming andexpensive robotics to deposit or synthesize nucleic acids atpredetermined locations.

A detecting apparatus consistent with the present invention may be usedas part of a system for or in methods and processes of biomoleculedetection, including nucleic acid hybridization or sequencing for, e.g.,whole genome sequencing, transcriptional profiling, comparativetranscriptional profiling, or gene identification. Biomolecule detectioncan also include detection and/or measurement of binding interactions,e.g., protein/protein, antibody/antigen, receptor/ligand, and nucleicacid/protein. These applications are useful for analytical or diagnosticprocesses and methods.

Nucleic acids suitable for detection on the apparatus provided by theinvention may, in some embodiments, be part of a linking molecule, whichaffixes a molecule suitable for assaying binding interactions, e.g.,proteins, other nucleic acids, carbohydrate moieties, or small moleculesto a linker site on a device provided by the invention. The linkingmolecule may, in some embodiments, further comprise a capture molecule,which binds to the molecule being assayed for binding interactions. Thenucleic acid in a linking molecule serves as an identifying tag for thecapture molecule of the linking molecule by, e.g., direct sequencing orhybridization.

The methods provided by the invention may comprise a step of affixing amolecule to be detected to an address array of a detecting systemprovided by the invention. In some embodiments, the address array mayinclude a blind sheet having a plurality of pinholes, and linker sitesmay be formed in or around the pinholes. Thus, a detecting systemconsistent with the present invention may simultaneously read millionsof nucleic acid segments. If each segment is, for example, 1000 baseslong, a single device could obtain billions of bits of sequenceinformation, making, e.g., whole genome sequencing and resequencingpossible.

3.1.1 Molecules to be Detected

Nucleic acids suitable for detection by the methods provided by thepresent invention may include any nucleic acid, including, for example,DNA, RNA, or PNA (peptide nucleic acid), and may contain anysequence—both known and unknown, including naturally occurring orartificial sequences. The nucleic acid may be naturally derived,recombinantly produced, or chemically synthesized. The nucleic acid maycomprise naturally-occurring nucleotides, nucleotide analogs notexisting in nature, or modified nucleotides. The length of the nucleicacid to be detected may vary based on the actual application. In someembodiments, the nucleic acid may include at least 10, 20, 50, 100, 200,500, 1000, 2000, 5000, 10000, 20000 bases, or more. In some embodiments,the nucleic acid may be from 10 to 20, from 10 to 50, from 10 to 100,from 50 to 100, from 50 to 500, from 50 to 1000, from 50 to 5000, from500 to 2000, from 500 to 5000, or from 1000 to 5000 bases.

A nucleic acid may be single-stranded for detection. Single strandednucleic acid templates may be derived from a double stranded molecule bymeans known in the art including, for example, heating or alkali orother chemical treatment. Single stranded nucleic acid templates mayalso be produced by, e.g., chemical or in vitro synthesis.

In some embodiments, the nucleic acid to be detected may be attached toa linker site at its 5′ or 3′ end. In some embodiments, the nucleic acidmay further comprise one or more end link primers coupled to the 5′ end,the 3′ end, or both the 5′ end and the 3′ end of the nucleic acid. Inparticular embodiments, an end link primer may be affixed to the 3′ endof the nucleic acid. End link primers may be used both to affix thenucleic acid to be detected to linker sites on the device and provide acomplementary sequence for one or more detecting primers, e.g., asequencing primer.

3.1.1.1 End Link Primer

End link primers are short nucleic acid molecules usually composed ofless than 100 nucleotides. In some embodiments, the end link primer maybe at least 5, 10, 15, 20, 25, 30, 50, 75, 90 nucleotides, or more, inlength. In certain embodiments, end link primers may be from 8 to 25,from 10 to 20, from 10 to 30, or from 10 to 50 nucleotides in length. Insome embodiments, the end link primers may be unbranched, however, inother embodiments, they may be branched.

The end link primer may be used to attach the nucleic acid to bedetected to a linker site on the address array. In some embodiments, theend link primer may link the nucleic acid to the array surface directly,e.g., by covalent linkage (e.g., ester or thiol linkage) or non-covalentlinkage, e.g., antigen/antibody or biotin/avidin binding. In someembodiments, the end link primer may link the nucleic acid to the arraysurface indirectly, e.g., by binding an intermediate molecule, e.g., apolymerase. Accordingly, the end link primer may contain modifiednucleotides or be otherwise modified to facilitate attachment to alinker site by means known in the art, e.g., disulfide, thioester,amide, phosphodiester, or ester linkages; or by, e.g., antibody/antigenor biotin/avidin binding, e.g., the end link primer may contain anucleotide comprising an antigen moiety or a biotinylated nucleotide. Inparticular embodiments, a modified nucleotide may be on the 3′ end of anend link primer. In some embodiments, the 5′ end of an end link primermay contain a modified nucleotide.

The end link primer may also serve as a complement to one or moreprimers used to detect the nucleic acid, e.g., a sequencing primer. Insome embodiments, the primer may be used to detect the nucleic acid byhybridization, e.g., the primer may contain a detectable label, e.g., afluorescent label. In some embodiments, the 5′ end of the end linkprimer may comprise a sequence complementary to a sequencing primer. Insome embodiments, the end link primer sequence that is complementary tothe sequencing primer may be oriented so that the 3′ end of thesequencing primer may be immediately adjacent to the first nucleotide inthe nucleic acid to be sequenced.

In some embodiments, end link primers may be added to ends of thenucleic acid to be detected by a ligase, for example, a DNA ligase. Insome embodiments, the end link primer and nucleic acid to be detectedmay be both single stranded before the ligation. In other embodiments,both may be double stranded. In still other embodiments, one may besingle stranded and the other may be double stranded. Ligation is wellknown in the art. For example, in the polony sequencing method, Shendureet al. (Science, 309:1728-1732 (2005)) ligated a T30 end link primer (32bp) to a sample DNA segment with the New England Biolabs' (NEB) QuickLigation kit. There, the ligation reaction solution included 0.26 pMoleof DNA, 0.8 pMole of T30 end link primer, 4.0 μl T4 DNA Ligase, in 1×Quick Ligation Buffer. After mixing, the reaction solution was incubatedfor about 10 minutes at room temperature, and then placed on ice. Theligation reaction was stopped by heating the samples to 65° C. for 10minutes.

In other embodiments, the end link primer may be synthesized on thenucleic acid to be detected. For example, the end link primer may be ahomopolymer added by, e.g., terminal transferase. For example, Harris etal., (Science 320:106-109 (2008)) added a poly A tail to DNA templates,which served as the complement to a poly T sequencing primer in thesingle molecule sequencing of a viral genome.

3.1.1.2 Sequencing Primer

A sequencing primer is a single-stranded oligonucleotide complementaryto a segment of the nucleic acid to be detected or its associated endlink primer. In some embodiments, the sequencing primer may be at least8, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides, or more in length. Inparticular embodiments, the sequencing primer may be from 8 to 25, from10 to 20, from 10 to 30, or from 10 to 50 nucleotides in length. Thesequencing primer may be made up of any type of nucleotide, includingnaturally-occurring nucleotides, nucleotide analogs not existing innature, or modified nucleotides. In certain embodiments, the 5′-end of asequencing primer may be modified to facilitate binding to a linker siteon the address array after the sequencing primer hybridizes with anucleic acid to be sequenced, including one or more end link molecules.

In some embodiments, a sequencing primer may contain modifiednucleotides, e.g., locked nucleic acids (LNAs; modified ribonucleotides,which provide enhanced base stacking interactions in a polynucleicacid). As an illustration of the utility of LNAs, Levin et al. (NucleicAcid Research 34(20):142 (2006)) showed that a LNA-containing primer hadimproved specificity and exhibited stronger binding relative to thecorresponding unlocked primer. Three variants of the MCP1 primer(5′-cttaaattttcttgaat-3′) containing 3 LNA nucleotides (in caps) atdifferent positions in the primer were made:MCP1-LNA-3′(5′-cttaaattttCtTgaAt-3′);MCP1-LNA-5′(5′-CtTaAattttcttgaat-3′); and MCP1-LNA-even(5′-ctTaaatTttctTgaat-3′). All LNA-substituted primers had enhanced Tm,while the MCP1-LNA-5′ primer exhibited particularly enhanced sequencingaccuracy (Phred Q30 counts). Accordingly, in particular embodiments, thesequencing primer may contain at least one locked nucleotide in its 5′region, i.e., the 5′ half, third, or quarter of the sequencing primer.

Sequencing primers and single stranded sample nucleic acids (i.e., anucleic acid to be detected including at least one end link primer) maybe hybridized before being applied to a detecting apparatus consistentwith the present invention. The sequencing primer and sample nucleicacid may be hybridized by mixing the sample nucleic acid with a molarexcess of sequencing primer in a salt-containing solution, such as 5×SSC(or 5×SSPE), 0.1% Tween 20 (or 0.1% SDS), and 0.1% BSA buffer. Themixture may be heated to 65° C. for at least 5 minutes and slowly cooledto room temperature, to allow primer/template annealing. Residualprimers may be eliminated by appropriate means including, e.g., amolecular sieve.

Primers, including both end link and sequencing primers, may be designedby appropriate means, including visual inspection of the sequence orcomputer-assisted primer design. Numerous software packages areavailable to assist in the primer design, including DNAStar™ (DNAStar,Inc., Madison, Wis.), OLIGO 4.0 (National Biosciences, Inc.), VectorNTI® (Invitrogen), Primer Premier 5 (Premierbiosoft), and Primer3(Whitehead Institute for Biomedical Research, Cambridge, Mass.). Primersmay be designed taking into account, for example, the molecule to besequenced, specificity, length, desired melting temperature, secondarystructure, primer dimers, GC content, pH and ionic strength of thebuffer solution, and the enzyme used (i.e., polymerase or ligase). See,e.g., Joseph Sambrook and David Russell, Molecular Cloning: A LaboratoryManual Cold Spring Harbor Laboratory Press; 3rd edition (2001).

3.1.1.3 Bonding to the Array Surface

After the sequencing primer and nucleic acid to be sequenced, includingone or more end link primers, are annealed, this complex may be preparedin a suitable buffer, applied to the surface of an address array, andallowed to bind. In some embodiments the sample nucleic acid (nucleicacid to be detected and one or more end link primers) may be affixed tolinker sites and sequencing or detecting primers may be later applied.In other embodiments, the complex may be hybridized before being appliedto a device. Linker sites where only one sample nucleic acid is boundare known as effective addresses. In certain embodiments, the complexmay be applied to the detecting system and the sample nucleic acidsaffixed to random linker sites on the address array. In otherembodiments, sample nucleic acids may be applied to predetermined linkersites on the address array by appropriate means, including, e.g., byrobotics or liquid handling systems.

Appropriate means for affixing nucleic acids to a solid support are wellknown in the art. In some embodiments, the sample nucleic acid may beaffixed directly to a linker site by covalent linkage, e.g., disulfide,thioester, amide, phosphodiester, or ester linkages; or by non-covalentlinkage, e.g, antibody/antigen or biotin/avidin binding. In someembodiments, the sample nucleic acid may be affixed to a linker site byan intervening molecule. In some embodiments, the intervening moleculemay be a polymerase, e.g., a DNA polymerase.

As an illustrative example of direct, covalent attachment of a nucleicacid, Adeesi et al. (Nucleic Acid Research, 28:87 (2000)) modified the5′ end of a primer to include a SH functional group. According to themethod of Adeesi et al., a nucleic acid may be prepared in 50 μMphosphate buffered saline (“PBS”) (NaPi: 0.1 MNaH₂PO₄ pH 6.5, 0.1 MNaCl). About 1-5 μl of primer solution may then be applied to a surfaceof a silanised glass slide and incubated in a humidity control box atroom temperature for about 5 hours to bond the primer to the chipsurface. After the binding reaction is completed, the PBS solution isvibration washed twice at room temperature for 5 minutes each to removeun-bonded DNA. After cleaning, 10 mM β-mercaptoethanol is added to a PBSsolution and used to rinse the address array surface under roomtemperature, to deactivate the thiol group of un-bonded DNA. Next, thearray surface is washed, e.g., once with 5×SSC 0.1% Tween and once with5×SSC buffer solution. Accordingly, in some embodiments, the method usedby Adeesi et al. can be used in the methods provided by the invention toaffix the sample nucleic acid complex to a linker site, e.g., via the 5′end of a sequencing primer or the sample nucleic acid.

In an alternative embodiment, the sample nucleic acid may comprise,e.g., a biotinylated nucleotide, and binds to avidin on the linker sitesurface. In another embodiment, the sample nucleic acid may comprise anantigenic moiety, e.g., BrdU or digoxigenin, that is bound by anantibody (or fragment thereof) on the linker site. By “antibody” it isto be understood that this term includes fragments of immunoglobinmolecules, including, for example, one or more CDR domains; or variableheavy or variable light fragments. Antibodies may be naturallyoccurring, recombinant, or synthetic. Antibodies may also include, e.g.,polyclonal and monoclonal variants. In some embodiments the antibodiesmay bind their antigen(s) with association constants of at least 10⁸,10⁷, 10⁸, 10⁹ M, or higher. The structure, function, and production ofantibodies are well known in the art. See, for example, Gary Howard andMatthew Kasser, Making and Using Antibodies: A Practical Handbook CRCPress; 1^(st) edition (2006).

In yet another embodiment, the sample nucleic acid may be affixed to thelinker site by a polymerase, e.g., DNA polymerase. The skilled artisanwill appreciate, that to retain enzyme function available information,such as the primary, secondary, and tertiary structures of the enzyme,should be taken into consideration. For example, the structures of Taqand Phi29 polymerases are known in the art, see: Kim et al., Nature,376:612-616 (1995) and Kamtekar et al., Mol. Cell, 16:609-618 (2004),respectively. Means for fixing a polymerase to a surface, whileretaining activity are known in the art and are described in, e.g., U.S.Patent Application Publication No. 2008/0199932, published Aug. 21, 2008and Korlach et al. PNAS 105:1176-1181 (2008).

In some embodiments, an aldehyde-modified surface of a linker site istreated with aldehyde-containing silane reagent. The aldehydes readilyreact with primary amines on the proteins to form a Schiff's baselinkage. Because many proteins display lysines on their surfaces inaddition to the generally more reactive α-amine at the NH₂-terminus,they may attach to the slide in a variety of orientations, permittingdifferent sides of the protein to interact with other proteins or smallmolecules in solution. In another embodiment, a photoNHS (a N-hydroxysuccimido carboxylate molecule linked to an azidonitrobenzene moleculewith a carbon chain linker) may attach to an amine-modified surface onthe device by UV photoactivation. In these embodiments, UV light excitesthe azidonitrobenzene moiety to produce highly reactive nitrene, byeliminating nitrogen. Nitrene readily reacts with NH₂ on the surface ofthe device and forms a hydrazine bond. The other end of the linker isNHS carboxylate, which react with lysines on the surface of polymeraseto produce an amide covalent bond. In another embodiment, an NHScarboxylate moiety may be reacted with primary amine on the surface ofthe device under buffered conditions. UV light may be used to activatean azidonitrobenzene moiety and form a highly reactive nitrene as anelectron deficient group and readily react with primary amine of lysineresidues on the polymerase.

3.1.2 Sequencing Modalities

The detecting apparatuses and methods provided by the present inventionmay be used to detect and sequence nucleic acids by means known in theart, as reviewed in, e.g., U.S. Pat. No. 6,946,249 and Shendure et al.,Nat. Rev. Genet. 5:335-44 (2004). The sequence modalities can be chosenfrom single molecule sequencing methods known in the art. In someembodiments, the sequencing methods may rely on the specificity ofeither a DNA polymerase or DNA ligase and may include, e.g., baseextension sequencing (single base stepwise extensions), multi-basesequencing by synthesis (including, e.g., sequencing withterminally-labeled nucleotides), and wobble sequencing, which isligation-based. The methods typically involve providing a sample nucleicacid, which may include at least one end link primer. The nucleic acidcan be affixed to a substrate (either directly or indirectly), e.g., ata linker site. The nucleic acid may be provided in single stranded formor may be rendered single stranded, e.g., by chemical or thermaldenaturation. Sequencing may be then initiated at a sequencing primer(ligase-based sequencing commonly refers to anchor primers, which servethe analogous purpose to sequencing primers).

For single molecule sequencing modalities, the present invention canoffer the advantage of being able to resequence single molecules. Forexample, after completion of a sequencing read, the sequencing primerand extended nucleotides may be stripped from the sample nucleic acid,the device is washed, and the sequencing is repeated. In variousembodiments, the resequencing may be done by the same or differentmethods. By resequencing the same molecule, sequencing errors areexpected to fall as the power of the number of sequencing reads. Forexample, if per base error rates for a single read are 10⁻³, then aftertwo reads, this falls to (10⁻³)², i.e., 10⁻⁶. This is particularlyadvantageous for single molecule sequencing since the modifiednucleotides used for sequencing can lose their labels or blocking groupsresulting in, e.g., spurious deletions.

In general, in single molecule sequencing, at least one nucleic acidmolecule to be sequenced is affixed to a substrate and contacted with aprimer. The primer is modified, e.g., by performing at least oneenzyme-catalyzed polymerization or ligation reaction. The at least onereaction leads to emission of light correlated to the identity of atleast one base comprised by the nucleic acid. “Leading to” emission oflight is understood to mean that the at least one reaction causes atleast one condition under which light emission correlated to theidentity of at least one base comprised by the nucleic acid occurs; thisoccurrence may be via interaction with excitatory light, a chemi- orbio-luminescent system, etc. The at least one condition can be, forexample, incorporation of a fluorophore into the product of the at leastone reaction, or the release of pyrophosphate. Thus, light may begenerated with or without external excitation. For example, singlemolecule sequencing can be performed with reversible terminator baseanalogs comprising a covalently-linked detectable label, e.g., afluorescent label, and a blocking group to prevent any secondaryextension, wherein the analog is excited and detected after it has beenadded to the primer, and the label and blocking group are removed afteraddition to allow another round of extension. Alternatively, a productof an extension step, such as a pyrophosphate, can be detected withoutexternal excitation by providing a chemi- or bio-luminescent detectionsystem which emits light in a pyrophosphate-dependent manner. These andother modalities are discussed in more detail below.

The light emitted is correlated to the identity of at least one basecomprised by the nucleic acid. In some embodiments, the correlation canbe temporal; e.g., the time of emission of the light indicates theidentity of the at least one base, such as is the case when differentbase analogs are provided for use in a polymerization reaction atdifferent times. In some embodiments, the correlation can be spectral;e.g., the spectrum of the emitted light indicates the identity of the atleast one base, such as is the case when different base analogs thatcomprise different fluorophores are provided for use in a polymerizationreaction.

In some embodiments, single molecule nucleic acid sequencing comprisesmultiple sequencing cycles. A sequencing cycle is understood to mean theevents that lead to an emission of light correlated to the identity ofat least one base that would be repeated in order to identify at least asecond base comprised by the nucleic acid after a first base has beenidentified. Thus, in methods according to the invention that comprisesingle molecule nucleic acid sequencing, the single molecule nucleicacid sequencing can comprise at least a given number of sequencingcycles that lead to at least the given number of emissions of lightcorrelated collectively to the identity of at least the given number ofbases comprised by the nucleic acid, and the method comprisesidentifying at least the given number of bases comprised by the nucleicacid. In some embodiments, the given number may be, for example, 2, 3,4, 5, 10, 20, 50, 100, 200, or 500.

Sequencing methods can comprise determining the identity of one or morebases comprised by a nucleic acid. In some embodiments of methodsaccording to the invention, in which performing single molecule nucleicacid sequencing leads to emission of light that is detected with atleast one light detector comprising at least a first optical sensor anda second optical sensor, and output signal from the at least two opticalsensors is processed, the identity of at least one base comprised by anucleic acid can be determined by comparing at least one result of theprocessing with at least one known result corresponding to at least oneknown type.

For example, a result of the processing can indicate a time at which areaction occurred; when light emitted is temporally correlated to theidentity of at least one base comprised by the nucleic acid, said timecan be used to identify at least one base comprised by the nucleic acid.

In another example, a result of the processing can be a determination ofwhich fluorophore was incorporated into the product of a reaction; whenlight emitted is spectrally correlated to the identity of at least onebase comprised by the nucleic acid, said determination can be used toidentify at least one base comprised by the nucleic acid.

3.1.2.1 Base Extension Sequencing: Stepwise Extension

In some embodiments, a detecting apparatus provided by the invention maybe used to detect light generated during base extension sequencing. Insome embodiments, base extension sequencing begins by attaching apartial duplex sample nucleic acid comprising a single stranded nucleicacid to be sequenced, an end link primer associated with the 3′ end ofnucleic acid to be sequenced, and a sequencing primer annealed thereto,to a linker site. In some embodiments, polymerase and modifiednucleotides may be then applied to the light detection apparatus in asuitable buffer. In some embodiments, the sample nucleic acid complexmay be affixed to the linker site by a polymerase at a linker site. Insome embodiments, the nucleotides may include a covalently-linkeddetectable label, e.g., a fluorescent label, and a blocking group toprevent any secondary extension. Accordingly, the sequencing pausesafter the addition of a single nucleotide to the 3′ end of sequencingprimer.

In a first step of one embodiment of a base extension sequencingreaction, a nucleotide with a fluorescent blocking group may be added bya DNA polymerase to the 3′ end of sequencing primer. In someembodiments, the fluorescent label may act as the blocking group. Inother embodiments, they may be separate moieties. A single nucleotidemay be incorporated at the 3′ end of sequencing primer and is identifiedby its label by the corresponding light detector. The fluorescent labeland blocking group are then removed, e.g., by chemical or enzymaticlysis, to permit additional cycles of base extension. In certainembodiments, the label and blocking groups may be removed simultaneouslyor sequentially and in any order. By compiling the order of the basesadded, the sequence of the sample nucleic acid may be deduced in the 3′to 5′ direction, one base at a time.

Generally, there are two ways to recognize the nucleotide added duringstepwise extension. In the first case, the four nucleotides may all havethe same detectable label, but are added one at a time, in apredetermined order. The identity of the extended nucleotide may bedetermined by the order that the nucleotide is added in the extensionreaction. In the second mode for recognizing the base integrated duringextension, four different nucleotides may be added at the same time andeach is coupled with a distinct detectable label. In differentembodiments, the excitation or emission spectra and/or intensity of thelabels may differ. The identity of the nucleotide added in the extensionmay be determined by the intensity and/or wavelength (i.e., excitationor emission spectra) of the detected label.

3.1.2.2 Sequencing by Synthesis: Multi-Step Extension

In some embodiments, sequencing by synthesis may proceed with multipleuninterrupted extensions, e.g., without the use of blocking groups. Inthese embodiments, the polymerization reaction may be monitored bydetecting the release of the pyrophosphate after nucleoside triphosphatehydrolysis, i.e., the release of the β and y phosphate complex. Thiscomplex may be detected directly, for example, by a fluorescent moietyon the complex, or indirectly, for example, by coupling thepyrophosphate to a chemi- or bio-luminescent detection system, asdiscussed above.

In some embodiments, the sample nucleic acid may be sequencedessentially continuously by using terminal-phosphate-labelednucleotides. Exemplary embodiments of terminal-phosphate-labelednucleotides and methods of their use are described in, e.g., U.S. Pat.No. 7,361,466 and U.S. Patent Publication No. 2007/0141598, publishedJun. 21, 2007. Briefly, the nucleotides may be applied to the systemprovided by the invention and, when hydrolyzed during thepolymerization, the labeled pyrophosphate may be detected by acorresponding light detector. In some embodiments, all four nucleotidesmay comprise distinct labels and be added simultaneously. In someembodiments, the nucleotides may comprise indistinguishable, e.g.,identical, labels and be added sequentially in a predetermined order.Sequential, cyclical addition of nucleotides with indistinguishablelabels still permits multiple, uninterrupted polymerization steps, e.g.,in homopolymer sequences.

3.1.2.3 Ligase-Based Sequencing

In other embodiments, a sample nucleic acid may be sequenced on theapparatus provided by the invention by ligase-based sequencing.Ligase-based sequencing methods are disclosed in, for example, U.S. Pat.No. 5,750,341, PCT publication WO 06/073504, and Shendure et al.Science, 309:1728-1732 (2005). In the method of Shendure et al., forexample, an unknown single-stranded DNA sample may be flanked by two endlink primers and immobilized on a solid support. A particular positionin the unknown sequence (e.g., the n^(th) base proximal to a particularend link primer) can be interrogated by annealing a so-called anchorprimer (which is analogous to a sequencing primer) to one of the endlink primers and then applying a pool of 4 degenerate nonamers to themixture. The four nonamers all have distinct fluorescent labels and aredegenerate at all positions except for the query position, where eachnonamer interrogates with a distinct base—A, C, G, or T. The sample iswashed, fluorescently scanned, and the query base is identified. Theanchor primer and ligated nonamer are then stripped from the samplenucleic acid, the device is washed, and the process is repeated,querying a different position. Advantageously, this method isnon-progressive, i.e., bases need not be queried in order. Thus, errorsare not cumulative. Additionally, this method can query nucleotides fromeither the 5′ or 3′ direction, i.e., does not require canonical 5′→43′DNA synthesis. A total of about 13 bases of a sample nucleic acid cantypically be sequenced by this method.

3.1.2.4 Third-Generation Sequencing

In some embodiments, a sample nucleic acid may be sequenced on theapparatus provided by the invention using third-generation sequencing.In third-generation sequencing, a slide with an aluminum coating withmany small (˜50 nm) holes is used as a zero mode waveguide (see, e.g.,Levene et al., Science 299, 682-686 (2003)). The aluminum surface isprotected from attachment of DNA polymerase by polyphosphonatechemistry, e.g., polyvinylphosphonate chemistry (see, e.g., Korlach etal., Proc Natl Acad Sci USA 105, 1176-1181 (2008)). This results inpreferential attachment of the DNA polymerase molecules to the exposedsilica in the holes of the aluminum coating. This setup allowsevanescent wave phenomena to be used to reduce fluorescence background,allowing the use of higher concentrations of fluorescently labeleddNTPs. The fluorophore is attached to the terminal phosphate of thedNTPs, such that fluorescence is released upon incorporation of thedNTP, but the fluorophore does not remain attached to the newlyincorporated nucleotide, meaning that the complex is immediately readyfor another round of incorporation. By this method, incorporation ofdNTPs into an individual primer-template complexes present in the holesof the aluminum coating can be detected. See, e.g., Eid et al., Science323, 133-138 (2009). Use of the detecting system consistent with thepresent invention may provide high sensitivity, allowing more efficientdetection of incorporated dNTPs, resulting in relatively low error ratesand/or longer reads of interpretable sequence data.

3.1.3 Additional Applications

A detecting system consistent with the present invention maysimultaneously detect millions of nucleic acid segments. If each segmentis, for example, 1000 bases long, a single device could obtain upwardsof billions of base sequences at once. Discussed below are additionalapplications of the apparatuses and methods provided herein.

3.1.3.1 Whole Genome Sequencing

A detecting system consistent with the present invention may be used toperform whole or partial genome sequencing of, e.g., a virus, bacterium,fungi, eukaryote, or vertebrate, e.g., a mammal, e.g., a human.

Genomic DNA may be sheared into fragments of at least 20, 50, 100, 200,300, 500, 800, 1200, 1500 nucleotides, or longer, for sequencing. Insome embodiments, the sheared genomic DNA may be from 20 to 50, from 20to 100, from 20 to 500, from 20 to 1000, from 500 to 1200, or from 500to 1500 nucleotides long. In some embodiments, the nucleic acids to besequenced, along with associated end link primers, may be made singlestranded, annealed to a sequencing primer, and applied to a systemprovided by the invention for sequencing as described above.

3.1.3.2 Gene Expression Profiling

In other embodiments, a detecting system consistent with the presentinvention may be used to sequence cDNA for gene expression profiling.For example, mRNA levels may be quantified by measuring the relativefrequency that a particular sequence is detected on a device. Severalmillion cDNA molecules may be sequenced in parallel on a device providedby the invention. If a cell contains, on average, 350,000 mRNAmolecules, a transcript present at even one copy per cell is expected tobe sequenced approximately three times in one million sequencingreactions. Accordingly, the devices provided by the invention aresuitable for single molecule sequencing with single copy numbersensitivity.

cDNA synthesis is well known in the art and typically includes total RNAextraction with optional enrichment of mRNA. cDNA is produced from mRNAby steps including, for example: reverse transcription, for first strandsynthesis; RNAse treatment, to remove residual RNA; random hexamerpriming of the first strand, and second strand synthesis by DNApolymerase. The resultant cDNA is suitable for sequencing on the devicesprovided by the invention. Methods of isolating and preparing both DNAand RNA are well known in the art. See, for example, Joseph Sambrook andDavid Russell, Molecular Cloning: A Laboratory Manual Cold Spring HarborLaboratory Press; 3rd edition (2001).

In some embodiments, cDNA may be ligated with adapter poly nucleicacids, the adapters may be processed with specialized restrictionenzymes, and finally, the processed nucleic acids bind to complementaryoligonucleotides affixed at linker sites of an apparatus provided by theinvention. In particular embodiments, the adapter molecules may be endlink primers.

In some embodiments consistent with the present invention, the poly-Atail of an mRNA may serve as a suitable end link primer, which iscomplementary to a poly T sequencing primer.

3.1.3.3 Detecting and/or Measuring Binding Interactions

In other embodiments, a detecting apparatus may be used to detectvarious binding interactions including, e.g., DNA/DNA, RNA/RNA, orDNA/RNA base pairings, nucleic acid/protein interactions,antigen/antibody, receptor/ligand binding, and enzyme/substrate binding.In general, a sample molecule may be affixed to a linking molecule thatcomprises an identifying nucleic acid tag (ID). In some embodiments, thelinking molecule may further comprise a capture molecule that binds thesample molecule. The linking molecule may also comprise a means forbinding to a linker site; e.g., a moiety to facilitate covalent chemicallinkage, such as disulfide, thioester, amide, phosphodiester, or esterlinkages; or by non-covalent linkage, e.g., antibody/antigen orbiotin/avidin binding. In some embodiments, a linking molecule may beaffixed to the array by the ID tag.

A sample molecule may be applied to a system consistent with the presentinvention and affixed to a random linker site by its linker molecule,e.g., by binding a capture molecule located on the linking molecule. Insome embodiments, the sample molecule and linker molecules may be mixed,allowed to bind, and then applied to a device provided by the invention.In some embodiments, the linker molecule may be first applied to thedevice, allowed to affix to a linker site, and then the sample moleculemay be applied. Next, the ID may be detected (e.g., by hybridization orsequencing) by the methods consistent with the invention to identify theassociated sample molecule. A plurality of sample molecule species maybe affixed to the same array and may be distinguished by their labelwhile their binding interactions may be characterized using the uniqueIDs of the capture molecule it binds to. Thus, in some embodiments, amethod of detecting a labeled sample molecule may comprise the steps oflinking a sample molecule to a linker site of a system consistent withthe present invention by a linker molecule comprising a nucleic acid tag(ID), performing nucleic acid sequencing of the ID, and detecting thelabeled sample molecule. In particular embodiments, the nucleic acidsequencing may be base extension sequencing. In some embodiments thenucleic acid sequencing may be chosen from ligase-based sequencing, orterminal-phosphate-labeled nucleotide sequencing.

By using nucleotide “bits,” up to 4^(n) distinct capture molecules maybe affixed and identified on a detecting system consistent with thepresent invention, where n is natural number representing the length ofthe ID sequenced. For example, 5 nucleotides could provide over athousand unique IDs, while 12 nucleotides provide over 16 millioncombinations. For example, linker molecules may be affixed to a systemconsistent with the present invention and their locations may bedetermined by detecting their corresponding ID tag. The linker moleculesthen may serve as probes to, e.g., investigate binding interactions withone or more labeled sample molecules. That is, a system with one or morelinker molecules affixed to it may serve as a probe array.

In certain embodiments, the labeled sample molecules may befluorescently labeled. When bound to the capture molecule of a linkermolecule, a labeled sample molecule may be detected by the lightdetector corresponding to the linker site where the linker molecule isaffixed. Accordingly, in some embodiments, methods consistent with thepresent invention may further comprise the steps of applying a labeledsample molecule to a system consistent with the present invention anddetecting the labeled sample molecule. In particular embodiments, thesystem may have linker molecules comprising a nucleic acid tag (ID)affixed to its linker sites. Multiple labeled sample molecules may beapplied to a probe array simultaneously and be differentiated by theirlabels, e.g., by the intensity and/or wavelength of their fluorescentlabels. Dissociation constants for binding interactions between samplemolecules and labeled query molecules may be inferred based on bothkinetics (e.g., rates of docking/undocking) and statistics (e.g., theportions of sample molecules in the bound or unbound state at any giventime) at a given concentration of a labeled query molecule.

In some embodiments, the ID of a linking molecule may be at least 5, 10,15, 20, 25, 30, 40, 50, 75, 90, 100, 150, 200, or more, nucleotideslong. In some embodiments, the ID may be from 5 to 10, 20, 40, 80, or160; or from 10 to 20 or 50; or from 20 to 35 nucleotides long. The IDcontains a unique nucleic acid sequence, i.e., a nucleic acid to bedetected. In particular embodiments, the unique nucleic acid sequencemay be at least 1, 2, 4, 6, 8, 10, 12, 14, 16, 20, 24, 30, or morenucleotides long. In some embodiments, the unique nucleic acid sequencemay be from 4 to 10, 12, 15, or 20; or from 10 to 20 nucleotides long.The ID may comprise at least one end link primer, i.e., the ID maycontain a sequence complementary to a sequencing primer, which, in someembodiments, may be modified to attach to a linker site, e.g., bycontaining a biotinylated nucleotide. In some embodiments, the end linkprimer portion of the ID may be 3′ to the unique nucleic acid sequence.In some embodiments, it may be 5′ to the unique nucleic acid sequence.In still other embodiments, end link primers may be present at both the3′ and 5′ ends of the unique nucleic acid sequence.

In certain embodiments, sample molecules and capture molecules maycomprise moieties chosen from a carbohydrate, lipid, protein, peptide,antigen, nucleic acid, hormone, small organic molecule (e.g., apharmaceutical), or vitamin moiety; or a combination thereof. Thesemoieties may be naturally-occurring (e.g., biochemically purified) orsynthetic (e.g., chemically synthesized or recombinantly produced).Additionally, these substrates may contain no, some, or all non-nativecomponents (e.g. non-natural amino acids, blocking or protecting groups,etc.). In particular embodiments, a sample molecule or capture moleculesmay be proteins, e.g., a growth factor, peptide antigen, antibody, orreceptor.

Various means for conjugating nucleic acids to linker molecules orlinker sites are known in the art, as reviewed in, e.g., U.S. PatentPublication No. 2004/0038331. The '331 publication discloses methods offorming protein oligonucleotide conjugates on a solid-phase support.U.S. Pat. No. 4,748,111 provides one example of conjugating a protein tothe 3′ end of a nucleic acid. There, terminal transferase is first usedto add a ribose residue to the 3′ portion of the molecule. A periodateoxidation reaction then generates a 3′ aldehyde group on the nucleicacid, which then forms a covalent bond with an amide group of a protein.When a protein is conjugated to the 3′ end of the ID, attachment to alinker site is via the 5′ end of the ID.

In some embodiments, a capture molecule, e.g., a protein, may be linkedto the 5′ end of an ID. In these embodiments, the 3′ end of the ID or 5′end of a sequencing primer may be used to affix capture molecule to alinker site. U.S. Pat. No. 6,013,434, for example, disclosesoligonucleotide-polyamide conjugates, where the connection is via the 5′end of the oligonucleotide. U.S. Pat. No. 6,197,513 discloses both PNAand DNA conjugates to molecules with carboxylic acid moieties, e.g.,proteins, via the 5′ end of the nucleic acid. The PNA and DNA moleculescontain arylamine or aminooxyacetyl moieties. U.S. Pat. No. 6,153,737discloses oligonucleotides containing at least one 2′ functionalizednucleoside, suitable for conjugating a variety of molecules to it.

3.1.3.4 Additional Detection Methods

(a) FRET

In some embodiments, a molecule may be detected on a detecting apparatusprovided by the invention by Förster resonance energy transfer (FRET),sometimes known as fluorescence resonance energy transfer. As is knownin the art, FRET occurs when an excited donor molecule non-radiativelytransfers energy to an acceptor molecule, which emits the energy,typically as light. FRET can help reduce background light by, e.g.,providing greater spectral separation between effective excitation andemission wavelengths for a molecule being detected. FRET is often usedto detect close molecular interactions since its efficiency decays asthe sixth power of the distance between donor and acceptor molecules.For example, Zhang et al. (Nature Materials 4:826-31 (2005)) detectednucleic acid hybridization by FRET. There, a biotinylated nucleic acidtarget was conjugated to an avidin-coated quantum dot donor, which thenexcited a Cy5-conjugated DNA probe. In some embodiments, a labeledcapture molecule and labeled sample molecule may form a donor/acceptor(or vice versa) pair for detection by FRET.

In some embodiments of nucleic acid sequencing provided by theinvention, fluorescently labeled nucleotides may act as acceptorchromophores for a donor chromophore attached to a polymerase or ligase.Accordingly, in these embodiments, the donor chromophore located on thepolymerase or ligase may excite an acceptor chromophore on a nucleotidebeing polymerized on, or ligated to, the sample nucleic acid.Nucleotides not proximate to the polymerase may be not excited due tothe rapid falloff in FRET efficiency. In some embodiments the donormolecule may be, e.g., another fluorophore, e.g., a quantum dot. Quantumdots, e.g., semiconductor quantum dots are known in the art and aredescribed in, e.g., International Publication No. WO 03/003015. Means ofcoupling quantum dots to, e.g., biomolecules are known in the art, asreviewed in, e.g., Medintz et al., Nature Materials 4:435-46 (2005) andU.S. Patent Publication Nos. 2006/0068506 and 2008/0087843, publishedMar. 30, 2006 and Apr. 17, 2008, respectively. In some embodiments,quantum dots may be conjugated to a DNA polymerase molecule. As alreadydiscussed above for conjugating enzymes to linker sites, the skilledartisan will undoubtedly appreciate that when conjugating fluorophoresto, e.g., a DNA polymerase or ligase, care must be taken to retainenzyme function by mitigating any effect of conjugating the fluorophoreon the primary, secondary, and tertiary structures of the enzyme.

(b) Multi Photon Excitation

In some embodiments, a chromophore may be excited by two or morephotons. For example, in some embodiments, excitation of either a donoror acceptor chromophore in FRET may be via two or more photons. Twophoton and multi-photon excitation are described further in, e.g., U.S.Pat. Nos. 6,344,653 and 5,034,613.

(c) Time Resolved Detection

In some embodiments, the excitation light source and light detectors ofan apparatus provided by the invention may be modulated to have acharacteristic phase shift. Using methods known in the art, for example,as disclosed in U.S. Patent Publication No. 2008/0037008, published Feb.14, 2008, light emitted from a molecule being detected on an apparatusprovided by the invention may be measured by a corresponding lightdetector without interference from an excitation light source.

(d) Flow Cytometry

In some embodiments, the excitation light source and light detectors ofan apparatus provided by the invention may be used to acquire flowcytometry data. Flow cytometry generally involves optical analysis of apopulation of objects in a liquid suspension. The suspension can beflowed past a detector, thereby allowing sequential detection of lightfrom many objects in the population. The objects can be chosen from, forexample, cells, microbeads, or other particles of similar sizes, such asparticles greater than 0.1 μm, 0.2 μm, 0.5 μm, 1 μm, 2 μm, or 5 μm in atleast one dimension (length, width, height, diameter, or the like, asappropriate for the shape of the particle), and/or smaller than 5 mm, 2mm, 1 mm, 500 μm, 200 μm, 100 μm, 50 μm, 20 μm, or 10 μm in at least onedimension or all dimensions. The objects can be passed single-filebetween one or more excitation light sources and detectors, andfluorescence and/or light scattering data can be acquired. In someembodiments, the objects can comprise at least one, at least two, atleast three, or at least four species of fluorophore. In someembodiments, light emitted by at least a subpopulation of the objects isdetected.

The fluorophore can be chosen from, for example, fluorophoresendogenously expressed by cells, such as fluorescent proteins (GFP, BFP,CFP, YFP, RFP, etc.), in addition to the fluorophores discussed above,including fluorophores with specific binding activity for a class ofbiomolecule or a cellular structure (e.g., DAPI, which is specific forDNA, or Evans Blue, which stains plasma membranes), fluorophoresconjugated to nucleotide analogs, e.g., cyanine-dye conjugated dUTP, andsynthetic fluorophores conjugated to a specific binding partner, such asan antibody, avidin, or a nucleic acid probe. The amount of fluorophorepresent in and/or bound to a particle, and therefore the amount ofemission the fluorophore generates when excited by light of anappropriate wavelength, can be correlated to the presence and/or amountof a biomolecule, such as DNA, plasma membrane, or a specific nucleicacid, protein, other biomolecule, or metabolite in or on the particle.

Flow cytometry can comprise analyzing a population of particles toproduce a frequency distribution of fluorescence intensities, such as ahistogram. When more than one fluorophore is used and/or lightscattering data is also acquired, or the data acquisition istime-resolved, the frequency distribution can be multidimensional. Useof a excitation light source and light detectors of an apparatusprovided by the invention may result in high quality data, for example,by allowing data acquisition with high sensitivity and/or a high signalto noise ratio.

In some embodiments, methods of the invention comprising performing flowcytometry can further comprise fluorescence activated sorting. In suchembodiments, particles are analyzed in real time by flow cytometry andsorted according to user-defined parameters. For example, particlesexhibiting detectable fluorescence from a given fluorophore, or thatexhibit such fluorescence within a given range, can be sorted apart fromparticles that do not meet the criterion. These particles can becollected for further analysis. In some embodiments, the particlessorted in this way are living cells. The sensitivity of detectionapparatuses according to the invention may allow the sorting of cellswith a low level of an activity, such as an enzyme activity or apromoter activity, apart from cells with undetectable activity and fromcells with high activity. Thus, the methods and apparatuses of theinvention may allow access to previously inaccessible enrichedpopulations of cells with a given low promoter or enzyme activity.

(e) Other Fluorescent Detection Apparatuses and Methods

In some embodiments, methods of the invention relate to detection oflight emitted by at least one object comprised by a biological cell,which can be a living or fixed cell. In some embodiments, the at leastone object is chosen from at least one object comprising at least onequantum dot, at least one object comprising at least one fluorescentprotein, and at least one object comprising at least one fluorescentsmall chemical moiety. In some embodiments, the at least one object isfluorescently labeled and comprises at least one oligonucleotide,polynucleotide, oligopeptide, polypeptide, oligosaccharide,polysaccharide, or lipid.

In some embodiments, the at least one object comprises a fixed andlimited number of fluorophores, such as at most 20, 10, 5, or 2fluorophores, which can be chosen from quantum dots, fluorescentproteins, and fluorescent small chemical moieties. In some embodiments,the at least one object comprises only a single fluorophore chosen froma quantum dot, a fluorescent protein, and a fluorescent small chemicalmoiety. Many examples of fluorescent small chemical moieties werediscussed above. In some embodiments, fluorescent small chemicalmoieties may have an emission peak between 300 and 800 nm and/or aquantum yield (fraction of photons emitted per photon of peak absorptionwavelength absorbed) of at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,or 0.9.

3.2 Biomolecule Analysis Service

The present invention also provides a method of providing biomoleculeanalysis service using a detecting apparatus in accordance withembodiments consistent with the present invention. In some embodiments,the method may include the steps of providing a sample including abiomolecule to be analyzed from a service requester to a serviceprovider and the service requester receiving analytical results from theservice provider, wherein the results may be produced using an apparatusprovided by the invention. In some embodiments, the method may beperformed for remunerative consideration, e.g., fee-for-service orcontract service agreements. In addition, the sample may be shippeddirectly between the service requester and the service provider, ormediated by a vendor. In some embodiments, the service provider orvendor may be geographically located in a territory outside of theUnited States of America, e.g. in another country.

4. Examples Example I

In this example, an apparatus as shown in FIG. 1 is used to detect anddiscriminate three types of objects. The light detector used in thisexample is a multi-junction photodiode with a P-N-P-N-P structure asshown in FIG. 17. P-type layer 601 and n-type layer 602 constitute thefirst optical sensor. P-type layer 603 and n-type layer 604 constitutethe second optical sensor. All three p-type layers are connected toground. The n-type layers 602 and 604 are connected to electrodes foroutputting signals J1 and J2, respectively.

A 6.5 mW laser diode with a center wavelength of 407 nm is used as theexcitation source to excite the object being detected. Light emittedfrom the object is incident on the light detector and is partiallyabsorbed by the first optical sensor and the second optical sensor,sequentially. FIGS. 18 and 19 show the responsivity curves of the firstoptical sensor and the second optical sensor, respectively.

The multi-junction photodiode is enclosed inside a black box to minimizethe impact of light from environment. A small hole is opened on thesurface of the box above the photodiode. The object being detected isinserted through the hole to a detection zone, where the object isexposed to the excitation light emitted from the excitation source. Along-pass interference thin-film optical filter may be placed betweenthe detection zone and the photodiode to partially block the scatteredexcitation light.

Three objects are used to test the detecting apparatus. Object 1 is afirst dye solution comprising Pyranine dissolved in deionized H₂O with aconcentration of 5.0×10⁻⁵M, Object 2 is a second dye solution comprisingRhodamine 6G (R6G) dissolved in deionized H₂O with a concentration of5.0×10⁻⁵M, and Object 3 is a quantum-dot solution comprising quantumdots with the composition of Cd_(x)Zn_(1-x)Se dissolved in Toluene witha concentration of 6.25×10⁻⁴M. FIGS. 20 and 21 show the absorptionspectra and emission spectra of the three objects, respectively.

In this example, a reference sample comprising water without dye orquantum dot is also tested. The objects and the reference sample aretested one at each time. The output signal J1 from the first opticalsensor and the output signal J2 from the second optical sensor and arerecorded every 5.12 milli-second. A record of 10 consequent readingsfrom each object and those from the reference sample is shown in Table1.

TABLE 1 Output signals from the light detector Object No object Object 1Object 2 Object 3 Signal J1 J2 J1 J2 J1 J2 J1 J2 1 0.0012 0.017 0.48611.036 0.1423 0.787 0.051 0.3807 2 0.0022 0.017 0.4866 1.036 0.1428 0.7870.0515 0.3807 3 0.0017 0.017 0.4851 1.036 0.1433 0.7865 0.051 0.3802 40.0017 0.017 0.4846 1.036 0.1418 0.7865 0.0505 0.3798 5 0.0007 0.01650.4866 1.036 0.1433 0.7865 0.0505 0.3807 6 0.0012 0.0165 0.4861 1.0360.1423 0.787 0.0505 0.3807 7 0.0022 0.016 0.4851 1.036 0.1423 0.7870.0501 0.3798 8 0.0022 0.016 0.4856 1.036 0.1428 0.787 0.0505 0.3812 90.0027 0.0165 0.4856 1.036 0.1438 0.7875 0.0501 0.3802 10  0.0022 0.01650.4851 1.036 0.1423 0.787 0.051 0.3798 Average 0.0020 0.017 0.486 1.0360.143 0.787 0.051 0.380 STD 0.0006 0.0004 0.0007 0.0000 0.0006 0.00030.0004 0.0005

In this example, a value of 0.2 is selected as the threshold value fordetermining whether an object is present. If any one of the outputsignals J1 and J2 is higher than 0.2, it is determined that an object ispresent.

After the presence of an object is determined, a ratio R=J2/(2×J1) iscalculated. If 0.5<R<1.5, it is determined that the object beingdetected is Object 1. If 2.0<R<3.0, it is determined that the objectbeing detected is Object 2. If 3.5<R<4.5, it is determined that theobject being detected is Object 3. Table 2 shows some examples ofdetecting results.

TABLE 2 Examples of detecting results Operation and ResultDiscrimination Test Conditions Presence Criteria Object 1 Object 2Object 3 Reading J1 J2 Type If J1 or J2 > 0.2 R = J2/(2 × J1) 0.5 < R <1.5 2.0 < R < 3.0 3.5 < R < 4.5 1 0.0017 0.0155 No object FALSE — — — —2 0.0032 0.0155 No object FALSE — — — — 3 0.4856 1.036 Object 1 TRUE1.067 TRUE FALSE FALSE 4 0.4841 1.036 Object 1 TRUE 1.070 TRUE FALSEFALSE 5 0.4851 1.036 Object 1 TRUE 1.068 TRUE FALSE FALSE 6 0.1423 0.787Object 2 TRUE 2.765 FALSE TRUE FALSE 7 0.1423 0.7855 Object 2 TRUE 2.760FALSE TRUE FALSE 8 0.0501 0.3749 Object 3 TRUE 3.742 FALSE FALSE TRUE 90.0476 0.3734 Object 3 TRUE 3.922 FALSE FALSE TRUE 10 0.0501 0.3739Object 3 TRUE 3.732 FALSE FALSE TRUE

Example II

Recent study showed that a defect in cytokine activation may occur inHCV/HIV co-infected persons that limit efficient clearance of HCV fromthe liver. (J. Med. Virol. 78:202-207, 2006). HCV and HIV viral RNA ofHCV mono-infected and HCV/HIV co-infected persons could be quantifiedusing a molecular beacon approach and detector of Example 1.

RNA Extraction

Total RNA can be extracted from liver biopsies of HCV monoinfected andHCV/HIV co-infected persons using the High Pure RNA Tissue kit (RocheDiagnostics; Meylan, France) and elute in 100 ul of RNase-free water.

cDNA Synthesis

The extracted total RNA of liver biopsies is reverse transcribed withthe Thermoscript Reverse Transcriptase kit using RC21 primer (5′-CTC CCGGGG CAC TCG CAA GC-3′ (SEQ ID NO:1)) for HCV and gagR primer(5′-TTTGGTCCTTGTCTTA TGTCCAGAATG-3′ (SEQ ID NO:2)) for HIV,respectively. The presence of any HCV or/and HIV RNA in the sample willincur reverse transcription and produce complementary single strandcDNAs. The RNA is then removed from the sample.

Molecular Beacon Probes

Different colors of molecular beacons are synthesized for detection ofHCV or

HIV. HCV: (5′-FAM-GCTAGCATTTGGGCGTGCCCCCGCIAGAGCTAGC-DABCYL-3′ (SEQ ID NO: 3)). HIV:(5′-HEX-GCTAGCATTTGGGCGTGCCCCCGCIAGAGCTAGC- DABCYL-3′ (SEQ ID NO: 4)).Detection of cDNA products in solution is carried out using MolecularBeacons (MBs) probes which was hybridized with their complementarysequences with denaturation step at 95° C. for 10 min, followed byannealing at 55° C. for 4 hours.Detection of the Targets

The hybridized cDNA sample is transported to an electrodeembeddedmicrofluidic reactor (Wang, T H et al., 2005) where a laser-focuseddetection region for single-molecule tracing is implemented. The samplesolution is introduced at the inlet of a microchannel and then driventhrough by hydrodynamic pumping. When molecules enter the electroderegion, their transport is governed by electrode-controlledelectrokinetic forces, which steer them toward the region of minimalenergy, located at the center of the middle electrode. The focused laserbeam of a confocal fluorescence spectroscope is positioned at thedownstream end of the energy minimum region, wherein fluorescent burstsemitted from individual molecules are detected with a detector ofExample 1. The presence of HCV and HIV in the sample are detected anddiscriminated from the dye labeled on the beacon probes using a methodas in Example I. The amount of HCV and HIV viral RNA in the sample isquantified according to the number of the detection reported by thedetecting apparatus.

For all patents, applications, or other reference cited herein, itshould be understood that it is incorporated by reference in itsentirety for all purposes as well as for the proposition that isrecited. Where any conflict exits between a document incorporated byreference and the present application, this application will dominate.

The specification is most thoroughly understood in light of theteachings of the references cited within the specification. Theembodiments within the specification provide an illustration ofembodiments of the invention and should not be construed to limit thescope of the invention. The skilled artisan readily recognizes that manyother embodiments are encompassed by the invention. All publications andpatents cited in this disclosure are incorporated by reference in theirentirety. To the extent the material incorporated by referencecontradicts or is inconsistent with this specification, thespecification will supersede any such material. The citation of anyreferences herein is not an admission that such references are prior artto the present invention.

Unless otherwise indicated, all numbers expressing quantities ofingredients, reaction conditions, and so forth used in thespecification, including claims, are to be understood as being modifiedin all instances by the term “about.” Accordingly, unless otherwiseindicated to the contrary, the numerical parameters are approximationsand may vary depending upon the desired properties sought to be obtainedby the present invention. At the very least, and not as an attempt tolimit the application of the doctrine of equivalents to the scope of theclaims, each numerical parameter should be construed in light of thenumber of significant digits and ordinary rounding approaches.

Unless otherwise indicated, the term “at least” preceding a series ofelements is to be understood to refer to every element in the series.Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed is:
 1. A method of sequencing a nucleic acid, comprisingthe steps of: (a) performing single molecule nucleic acid sequencing ofat least one nucleic acid molecule, wherein the single molecule nucleicacid sequencing leads to emission of light correlated to the identity ofat least one base comprised by the nucleic acid; (b) detecting the lightwith at least one light detector comprising a multi-junction photodiodewhich includes at least a first optical sensor configured to generate afirst output signal in response to the light and a second optical sensorconfigured to generate a second output signal in response to the light;wherein the first optical sensor comprises a first semiconductormaterial and the second optical sensor comprises a second semiconductormaterial different from the first semiconductor material, and the firstand second optical sensors have different responsive properties; (c)processing the first and second output signals by summing the first andsecond output signals to obtain a total signal, dividing the secondoutput signal by the total signal to obtain a first ratio, and dividingthe first output signal by the second output signal to obtain a secondratio; and (d) comparing the first ratio with a first known ratio rangecorresponding to a known type of nucleic acid base; (e) confirming theknown type of nucleic acid base by comparing the second ratio to asecond known ratio range corresponding to the known type of nucleic acidbase; and (f) determining the type of nucleic acid base when the firstratio corresponds to the first known ratio range and the second ratiocorresponds to the second known ratio range.
 2. The method of claim 1,wherein the first optical sensor includes a first p-n junction and thesecond optical sensor includes a second p-n junction.
 3. The method ofclaim 2, wherein the first p-n junction and second p-n junction arewithin one device.
 4. The method of claim 1, further comprisingcollecting and directing the light emitted from the nucleic acidmolecule to the optical sensors with an optical structure.
 5. The methodof claim 4, wherein the optical structure comprises a prism.
 6. Themethod of claim 4, wherein the optical structure comprises a grating. 7.The method of claim 4, wherein the optical structure comprises a lens.8. The method of claim 4, wherein the optical structure comprises anoptical filter.
 9. The method of claim 1, wherein the at least first andsecond optical sensors are stacked.
 10. The method of claim 1, whereinthe at least two optical sensors are arranged horizontally.
 11. Themethod of claim 1, wherein the light detector further comprises acircuit of signal amplifier, A/D convertor, integrator, comparator,logic circuit, readout circuit, memory, microprocessor, clock, and/oraddress.
 12. The method of claim 1, wherein the nucleic acid molecule islabeled with at least one object having light emitting capability. 13.The method of claim 12, wherein the object is chosen from at least onequantum dot, at least one fluorescent protein, and at least onefluorescent small chemical moiety.
 14. The method of claim 1, whereinthe nucleic acid molecule is fluorescently labeled.
 15. The method ofclaim 1, further comprising detecting a plurality of nucleic acidmolecules using the at least one light detector, wherein the pluralityof nucleic acid molecules are comprised by a liquid suspension, whichflows past the at least one light detector.